Detection of target nucleic acids in a cellular sample

ABSTRACT

Methods of assaying cells of a cellular sample for the presence of a target nucleic acid are provided. Aspects of the methods include evaluating a cellular sample that has been contacted with a nuclease inhibitor for the presence of a target nucleic acid. Also provided are devices and kits that find use in practicing the methods described herein.

CROSS-REFERENCE

This application is a continuation of U.S. patent application Ser. No.14/045,698 filed on Oct. 3, 2013, which application claims the benefitof U.S. Provisional Patent Application No. 61/709,896 filed on Oct. 4,2012; which applications are incorporated herein by reference in theirentirety.

GOVERNMENT RIGHTS

This invention was made with Government support under contract CA034233awarded by the National Institutes of Health. The Government has certainrights in the invention.

INTRODUCTION

Many disease conditions result from the aberrant behavior of individualcells, and such behavior is fundamentally the result of abnormalmolecules and/or aberrant molecular interactions. Molecular markers ofdisease therefore provide a foothold toward understanding the molecularcauses of aberrant cell behavior and facilitate the identification,quantification, and/or isolation of the aberrant cells. As such,molecular markers are an invaluable tool aiding in the early diagnosisof cancer and other debilitating conditions.

Flow cytometry, or fluorescent activated cell sorting (FACS), hasenabled the rapid analysis of molecular markers in individual cells of acell sample. In flow cytometry, cells of a cellular sample are suspendedin a stream of fluid, which is passed, one cell at a time, by at leastone beam of light (e.g., a laser light of a single wavelength). A numberof detectors, including one or more fluorescence detectors, detectscattered light as well as light emitted from the cellular sample (e.g.,fluorescence). In this way, the flow cytometer acquires data that can beused to derive information about the physical and chemical structure ofeach individual cell that passes through the beam(s) of light.

RNA has long been recognized as a critical class of molecular marker.Expression levels of specific RNAs are known to vary according to celltype, metabolic state, state of differentiation, state of activation orstimulation, etc. The advent of techniques for the assessment of RNAexpression levels for thousands of RNAs in parallel has led to thediscovery that the expression levels of many RNAs associate with variousmetabolic states, states of differentiation, states of stimulation,and/or disease states. Examples of such RNAs include SIRT1 (energyexpenditure and insulin sensitivity), CCL3 (inflammatory response),BACE1 and PP2A (Alzheimer's disease), KIF14 (lung cancer), and MDR1(chemotherapeutic response of primary breast tumors). Therefore, theevaluation of RNA expression in individual cells can be useful indiagnosis as well as prognosis.

SUMMARY

Methods of assaying cells of a cellular sample for the presence of atarget nucleic acid are provided. Aspects of the methods includeevaluating a cellular sample that has been contacted with a nucleaseinhibitor for the presence of a target nucleic acid. Also provided aredevices and kits that find use in practicing the methods describedherein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates a branched DNA amplification protocol according to anembodiment of the invention, which enables a broad range of cytometricRNA detection. (A) Schematic diagram depicting of one embodiment of thehybridization and amplification steps to detect a target nucleic acid.B. Genes expressed at a range of levels (GAPDH, PPIB, HPRT, POLR2A,HMBS) were detected by SCRF in U937 cells. C. SCRF had linearperformance in a broad dynamic range of concentration. Plotted on theY-axis are the average number of molecules/fluorescent signals per cells(calculated from microscopy images) and on the X-axis relative MFI(MFI_(gene)/MFI_(no probe)) D. Cells analyzed in B were examined byfluorescence microscopy. Blue indicates nuclear stain (DAPI). Greencorresponds to signal from branched DNA amplification of individualtranscripts.

FIG. 2 Co-expression of PU.1 and GATA1 in committed hematopoieticprogenitors. (A) lin⁻SCA1⁺cKIT⁺ cells were sorted live from mouse bonemarrow cells enriched by lineage depletion. PU.1 and GATA1 weresimultaneously detected by SCRF cytometry. (B) Populations marked withnumbered gates in Panel A were analyzed by microscopy. Projection ofconfocal Z-stacks are shown. Blue corresponds to nuclear membranestained with anti-lamin antibodies conjugated with Ax 488. Redcorresponds to GATA1 detected with antibody conjugated to Ax546. Yellowcorresponds to PU.1 detected with antibody conjugated to Ax647.

FIG. 3 Stochastic expression of IFNα by PDC. IFNα and TNF expressionwere measured at RNA and protein levels in unstimulated (A, B, C) FLDCsand in FLDCs stimulated for 11 hours in the presence of CpG (D, E, F).pDCs were identified as the B220^(high)120g8^(high) population (seegating in A and D). IFN and TNF protein and RNA levels weresimultaneously measured by SCRF in pDCs (B, E) and the rest of theculture (B220^(low) cells largely correspond to conventional DCs). InFIGS. 3G-I, IFNα and IRF7 RNA levels were measured by SCRF in pDCs frombone marrow activated for 11 hours in the presence of CpG.

FIG. 4 PU.1 and GATA expression in sorted subsets of lineage depletedmouse bone marrow. A. Population as defined by cKIT and SCA1 staining inlineage negative (lin−) bone marrow cells were enriched by magneticdepletion. B,C,D, Co-expression of PU.1 and GATA1 transcripts inlin−cKIT−, lin−SCA1+cKIT+ (LSK) and lin−SCA1-cKIT+ (LK) subsets sortedfrom lin− bone marrow and treated according to RNA flow protocol.

FIG. 5 Resilience of surface staining during the RNA flowhybridizations. Total mouse Bone Marrow was stained live with cocktailof biotinylated lineage antibodies (B220, TCRb, CD3, CD4, Ter119, CD19)followed by staining with streptavidin-Ax488 and CD34-Ax700. The cellswere then analyzed by flow cytometry, either immediately (A) afterfixation and permeabilization (B) or after first (C), second (D) andforth (E) RNA flow hybridizations. Rectangular gate marks CD34+lin−cells encompassing the population of hematopoietic progenitors. Note thepreservation of the CD34 and lineage staining in the course of theprotocol.

FIG. 6 Co-detection of GATA1 and PU.1 expression in lin− mouse bonemarrow subsets as defined by CD34 and Flt3 protein expression. A. lin−subsets defined by surface expression of SCA1 and cKIT protein epitopes.Lineage depleted mouse bone marrow cells were stained live with SCA1,cKIT, CD34 (B,C) or Flt3 (G-L) antibodies and further subjected to RNAflow protocol for the detection of PU.1 and GATA1 transcripts. B,C Gata1RNA positive cells of the LK and LSK subset do not express CD34. D,EGATA1 and PU.1 are expressed in mutually exclusive patterns in cKIT− andLK subsets of lin− cells. F GATA1 and PU.1 are co-expressed in LSKcells. G-I Flt3 high cells manifest broad range of PU.1 expression. J-LGATA1 transcript and Flt3 are expressed in mutually exclusive patternsin all subsets of lin− cells.

FIG. 7 Co-detection of GATA1 and PU.1 expression in lin− mouse bonemarrow subsets as defined by CD127 and FcgammaR protein expression. A-C.Expression of PU.1 transcript in cellular subsets defined by CD127levels. Majority of CD127 positive cells (containing the commonlydefined CLP) are in cKIT− population. Notably PU.1 high cells of LSKsubset (combining both myeloid and lymphoid potential) produce elevatedlevels of CD127, due to “priming” phenomenon. D-F. Expression of GATA1transcript in cellular subsets defined by CD127 levels. Except forco-expression in a GATA1 high subset of LSK cells GATA1 and CD127manifest mutually exclusive expression patterns. G-I. Expression of PU.1transcript in cellular subsets defined by FcgammaR levels. MajorityFcgammaR positive cells are in cKIT− population. FcgammaR high cells ofthe LK subset express high PU.1 matching their function as GMPs. J-L.Expression of GATA1 transcript in cellular subsets defined by FcgammaRlevels. GATA1 and FcgammaR manifest largely mutually exclusiveexpression patterns.

FIG. 8 Co-detection of C/EBPa (A-F) or GFI1B (G-L) with GATA1 (D-F, J-L)and PU.1 (A-C, G-I) expression in lin− mouse bone marrow subsets: cKIT−cells (A,D,G,J), LK cells (B,E,H,K) and LSK cells (C,F,I,L).

FIG. 9 Co-detection of GFI1 (A-F) or GATA2 (G-L) with GATA1 (D-F, J-L)and PU.1 (A-C, G-I) expression in lin− mouse bone marrow subsets: cKIT−cells (A,D,G,J), LK cells (B,E,H,K) and LSK cells (C,F,I,L).

FIG. 10 Co-detection of MEIS1, MYB and Runx1 with GATA1 and PU.1expression in lin− mouse bone marrow subsets. A-C. Co-expression ofMEIS1 and PU.1 in cKIT−, LK and LSK cells. D-F. Co-expression of MEIS1and GATA1 in cKIT−, LK and LSK cells. G. Background RNA flow signal inlin− cell subsets defined by cKIT levels (no primary probe was suppliedduring the first step of the RNA flow protocol, the rest of cascade wasapplied as in RNA flow with specific probes) H-I. Expression of Myb andRunx1 in lin− cell subsets defined by cKIT levels.

FIG. 11 Co-detection of E2A (A-F) or EBF1 (G-L) with GATA1 (D-F, J-L)and PU.1 (A-C, G-I) expression in lin− mouse bone marrow subsets: cKIT−cells (A,D,G,J), LK cells (B,E,H,K) and LSK cells (C,F,I,L).

FIG. 12 Co-detection of Pax5 (A-F) or Id2 (G-L) with GATA1 (D-F, J-L)and PU.1 (A-C, G-I) expression in lin− mouse bone marrow subsets: cKIT−cells (A,D,G,J), LK cells (B,E,H,K) and LSK cells (C,F,I,L).

FIG. 13 Postfixation allows preservation of RNA in fixed andpermeabilized cells (A) U937 cell line RNA was purified by Trizol fromfresh cells (right lane) or by RecoverALL kit (Ambion) from fixed,permeabilized and post-fixed cells (see protocol in Materials andMethods section) and were analyzed on an 1.5% agarose gel (lanes 1-3).Either 4% or 10% formaldehyde in PBST was used for post-fixation (middleand left lane). Skipping the postfixation step resulted in fulldegradation of RNA (not shown). (B) Live human monocytes (CD33+) weresorted from PBMCs of normal donor 1 (ND1) and ND2. RNA from live ND1monocytes was prepared using Trizol reagent. ND2 monocytes were fixed,permeablized and postfixed. RNA from ND2 monocytes was prepared byRecoverALL kit (Ambion). Microarray hybridization probes weresynthesized from total RNA using an Ovation Pico WTA kit (NuGEN) andwere biotin labeled using the Encore Biotin Module (NuGEN). Affymetrixmicroarrays U133 Plus2 were used to determine levels of gene expressionin RNA from live and fixed cells. High correlation between the geneexpression values in fixed and permeabilized ND2 (y-axis) versus live(x-axis) ND1 monocytes were observed by microarray screening.

FIG. 14 presents a schematic diagram depicting one embodiment of thehybridization steps to detect a target nucleic acid (e.g., a miRNA).

FIGS. 15A-B demonstrate successful detection of miRNAs in human U937cells using a method according to an embodiment.

FIG. 16 demonstrates successful detection of miRNA and proteinssimultaneously in the same cells using a method according to anembodiment.

DETAILED DESCRIPTION

Methods of assaying cells of a cellular sample for the presence of atarget nucleic acid are provided. Aspects of the methods includeevaluating a cellular sample that has been contacted with a nucleaseinhibitor for the presence of a target nucleic acid. Also provided aredevices and kits that find use in practicing the methods describedherein.

Before the present invention is described in greater detail, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. The invention encompassesvarious alternatives, modifications, and equivalents, as will beappreciated by those of skill in the art. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges and are also encompassed within the invention, subject toany specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Certain ranges are presented herein with numerical values being precededby the term “about.” The term “about” is used herein to provide literalsupport for the exact number that it precedes, as well as a number thatis near to or approximately the number that the term precedes. Indetermining whether a number is near to or approximately a specificallyrecited number, the near or approximating unrecited number may be anumber which, in the context in which it is presented, provides thesubstantial equivalent of the specifically recited number.

It is noted that, as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. It is further noted that the claimscan be drafted to exclude any optional element. As such, this statementis intended to serve as antecedent basis for use of such exclusiveterminology as “solely,” “only” and the like in connection with therecitation of claim elements, or use of a “negative” limitation.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which can be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentinvention. Any recited method can be carried out in the order of eventsrecited or in any other order which is logically possible.

Any publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference and are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present invention is not entitled to antedate suchpublication by virtue of prior invention. Further, the dates ofpublication provided may be different from the actual publication dateswhich may need to be independently confirmed.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, representativeillustrative methods and materials are now described.

Methods

Aspects of the invention include methods of assaying a cellular samplefor the presence of a target nucleic acid (e.g., deoxyribonucleic acid,ribonucleic acid). As such, methods of the invention are methods ofevaluating the amount (i.e., level) of a target nucleic acid in a cellof a cellular sample. In some embodiments, methods of the invention aremethods of evaluating whether a target nucleic acid is present in asample, where the detection of the target nucleic acid is qualitative.In some embodiments, methods of the invention are methods of evaluatingwhether a target nucleic acid is present in a sample, where thedetection of the target nucleic acid is quantitative. The methods caninclude determining a quantitative measure of the amount of a targetnucleic acid in a cell of a cellular sample. In some embodiments,quantifying the level of expression of a target nucleic acid includescomparing the level of expression of one nucleic acid to the level ofexpression of another nucleic acid in order to determine a relativelevel of expression. In some embodiments, the methods includedetermining whether a target nucleic acid is present above or below apredetermined threshold in a cell of a cellular sample. As such, whenthe detected signal is greater than a particular threshold (alsoreferred to as a “predetermined threshold”), the amount of targetnucleic acid of interest is present above the predetermined threshold inthe cell of a cellular sample. When the detected signal is weaker than apredetermined threshold, the amount of target nucleic acid of interestis present below the predetermined threshold in the cell of a cellularsample.

The term “cellular sample,” as used herein means any sample containingone or more individual cells in suspension at any desired concentration.For example, the cellular sample can contain 10¹¹ or less, 10¹⁰ or less,10⁹ or less, 10⁸ or less, 10⁷ or less, 10⁶ or less, 10⁵ or less, 10⁴ orless, 10³ or less, 500 or less, 100 or less, 10 or less, or one cell permilliliter. The sample can contain a known number of cells or an unknownnumber of cells. Suitable cells include eukaryotic cells (e.g.,mammalian cells) and/or prokaryotic cells (e.g., bacterial cells orarchaeal cells).

In practicing the methods of the invention, the cellular sample can beobtained from an in vitro source (e.g., a suspension of cells fromlaboratory cells grown in culture) or from and in vivo source (e.g., amammalian subject, a human subject, etc.). In some embodiments, thecellular sample is obtained from an in vitro source. In vitro sourcesinclude, but are not limited to, prokaryotic (e.g., bacterial, archaeal)cell cultures, environmental samples that contain prokaryotic and/oreukaryotic (e.g., mammalian, protest, fungal, etc.) cells, eukaryoticcell cultures (e.g., cultures of established cell lines, cultures ofknown or purchased cell lines, cultures of immortalized cell lines,cultures of primary cells, cultures of laboratory yeast, etc.), tissuecultures, and the like.

In some embodiments, the sample is obtained from an in vivo source andcan include samples obtained from tissues (e.g., cell suspension from atissue biopsy, cell suspension from a tissue sample, etc.) and/or bodyfluids (e.g., whole blood, fractionated blood, plasma, serum, saliva,lymphatic fluid, interstitial fluid, etc.). In some cases, cells,fluids, or tissues derived from a subject are cultured, stored, ormanipulated prior to evaluation. In vivo sources include livingmulti-cellular organisms and can yield non-diagnostic or diagnosticcellular samples.

Cellular samples can be obtained from a variety of different types ofsubjects. In some embodiments, a sample is from a subject within theclass mammalia, including e.g., the orders carnivore (e.g., dogs andcats), rodentia (e.g., mice, guinea pigs, and rats), lagomorpha (e.g.rabbits) and primates (e.g., humans, chimpanzees, and monkeys), and thelike. In certain embodiments, the animals or hosts, i.e., subjects (alsoreferred to herein as patients) are humans.

A target nucleic acid can be any polynucleotide nucleic acid molecule(e.g., DNA molecule; RNA molecule, modified nucleic acid, etc.). In someembodiments, the target nucleic acid is a coding RNA (e.g., mRNA). Insome embodiments, the target nucleic acid is a non-coding RNA (e.g.,tRNA, rRNA, microRNA (miRNA), mature miRNA, immature miRNA; etc). Insome embodiments, the target nucleic acid is an RNA molecule that is notnormally spliced (e.g., mRNA, rRNA, etc.) in the context of a cell. Insome embodiments, the target nucleic acid is an RNA molecule that isnormally spliced (e.g., mRNA, rRNA, etc.) in the context of a cell. Asuitable target nucleic acid can therefore be an unspliced RNA (e.g.,mRNA), a partially spliced RNA (e.g., an mRNA where one or more intronshave yet to be removed), or a fully spliced RNA (e.g., mRNA, “mature”mRNA), etc.

In some cases, the target sequence (i.e., the sequence with which aprobe hybridizes) is present in more than one species of the targetnucleic acid (e.g., the same sequence may be present in an unspliced, apartially spliced, and/or a fully spliced RNA). In such cases, thesubject methods can be used to simultaneously detect more than onespecies of the target nucleic acid. For example, in some embodiments,the subject methods can be used to detect a specific splice variant of atarget nucleic acid (e.g., mRNA) and/or a specific subset of splicevariants of the target nucleic acid.

In some cases, the target sequence within the target nucleic acid islocated at a splice junction of an mRNA molecule. In such cases, thesubject method can be used to detect spliced and/or partially splicedspecies (i.e., versions) of the target nucleic acid. For example, insome cases, the target sequence spans a splice junction such that thetarget sequence was generated by the process of mRNA splicing (e.g.,prior to splicing, the target sequence as a whole did not exist, and thesequences that make up the target sequence were brought together viasplicing). In some such cases, the target sequence is equallydistributed across the splice junction (e.g., each side of the splicejunction has the same (or similar) number of targeted nucleotides). Insome cases, the target sequence is non-equally distributed across thesplice junction (e.g., each side of the splice junction does not havesame number of targeted nucleotides). In some cases, one side of thesplice junction has all targeted nucleotides except for one nucleotide(the one nucleotide being introduced as the result of splicing).

In some cases, the target sequence is present in an unspliced RNA(and/or partially spliced RNA)(e.g., mRNA), but is removed by theprocess of splicing. In such cases, the subject method can be used todetect unspliced and/or partially spliced species (i.e., versions) ofthe target nucleic acid. For example, in some cases, the target sequencespans a future splice junction such that the target sequence isdestroyed by the process of mRNA splicing (e.g., prior to splicing, thetarget sequence exists, but the sequences that make up the targetsequence are removed and/or rearranged via splicing). In some suchcases, the target sequence is equally distributed across the futuresplice junction (e.g., each side of the future splice junction has thesame (or similar) number of targeted nucleotides). In some cases, thetarget sequence is non-equally distributed across the future splicejunction (e.g., each side of the future splice junction does not havesame number of targeted nucleotides). In some cases, one side of thefuture splice junction has all targeted nucleotides except for onenucleotide (the one nucleotide being removed as the result of splicing).

In some embodiments, the target nucleic acid is a splice variant. Theterm “splice variant” is used herein to mean a species (i.e.,version)(spliced, alternatively spliced, partially spliced, and/orunspliced version) of an RNA (e.g., an mRNA) that is not the mostprevalent species under normal circumstances. For example, in somecases, a splice variant is a fully spliced RNA (e.g., mRNA), but is avariant species (or species of low abundance or non-existent undernormal circumstances) (e.g., an alternatively spliced mRNA). In somecases, a splice variant is an unspliced or partially spliced RNA (e.g.,mRNA), and can be considered to be a species of low abundance undernormal circumstances. In some contexts, a splice variant may be the mostprevalent species of target nucleic acid (e.g., RNA) under a particularcontext (e.g., disease, altered metabolism, experimental manipulation,e.g., drug stimulation, change in temperature, change in oxygen, etc.),but is still considered a splice variant because the species is not themost prevalent species under normal circumstances.

In some embodiments, the target sequence spans the junction of a fusionmolecule (i.e., a fusion junction). For example, in some cases, a targetnucleic acid (e.g., DNA, mRNA) is a fusion molecule (i.e., two nucleicacid molecules that are normally separate molecules)(e.g., a fusion genetranscript). For example, a fusion molecule can be a fusion DNA or afusion mRNA molecule that codes for a fusion protein (two proteins thatare normally separate and normally coded for by separate mRNAmolecules). In some such cases, the target sequence spans the junctionbetween the normally separated nucleic acid molecules (i.e., the fusionjunction). In such cases, the subject methods can be used tospecifically detect a fusion nucleic acid molecule (e.g., where thetarget sequence is only present in the targeted fusion nucleic acidmolecule). For example, the subject methods can be used to detect DNAand/or RNA fusion molecules that result from disease-associated fusionevents (e.g., cancer-associated gene fusions), DNA and/or RNA fusionmolecules that result from human intervention (e.g., heterologousfusions such as DNA and/or mRNAs that encode fusion proteins, e.g.,proteins that may or may not be fused to a tag), etc.

Suitable target nucleic acid molecules include polynucleotides of a widerange of lengths. In some instances, a target nucleic acid molecule is10 or more, 15 or more, 20 or more, 25 or more, 30 or more, 35 or more,40 or more, 60 or more, 80 or more, 100 or more, 150 or more, 200 ormore, 300 or more, 400 or more, 500 or more, or 1,000 or more, 5,000 ormore, or 10,000 more nucleotides in length. In some instances, thetarget nucleic acid includes 10 or more consecutive nucleotides of knownsequence. For example, a target nucleic acid molecule can include 15 ormore, 20 or more, 25 or more, 30 or more, 35 or more, 40 or more, 60 ormore, 80 or more, 100 or more, 150 or more, 200 or more, 300 or more,400 or more, 500 or more, or 1,000 or more consecutive nucleotides ofknown sequence. In some instances, the sequence of the target nucleicacid molecule is unknown.

In some cases, the target nucleic acid (e.g., miRNA, mature miRNA, etc.)has a length in the range of from 10 nucleotide (nt) to 50 nt (e.g.,from 10 nt to 45 nt, from 10 nt to 40 nt, from 10 nt to 35 nt, from 10nt to 30 nt, from 10 nt to 25 nt, from 15 nt to 45 nt, from 15 nt to 40nt, from 15 nt to 35 nt, from 15 nt to 30 nt, from 15 nt to 25, or from17 nt to 23 nt).

In some cases, the target nucleic acid (e.g., immature miRNA, somemRNAs, etc.) has a length in the range of from 100 nt to 1500 nt (e.g.,from 100 nt to 1200 nt, from 100 nt to 1000 nt, from 100 nt to 800 nt,from 100 nt to 600 nt, from 100 nt to 400 nt, from 250 nt to 1500 nt,from 400 nt to 1500 nt, from 600 nt to 1500 nt, from 800 nt to 1500 nt,from 1000 nt to 1500 nt, from 1200 nt to 1500 nt, from 200 nt to 1400nt, from 300 nt to 1300 nt, from 500 nt to 1100 nt, from 600 nt to 1000nt, or from 700 nt to 1000 nt).

In some cases, the target sequence of a target nucleic acid (e.g.,miRNA, mature miRNA, splice junction, fusion junction, and the like) hasa length in the range of from 10 nt to 50 nt (e.g., from 10 nt to 45 nt,from 10 nt to 40 nt, from 10 nt to 35 nt, from 10 nt to 30 nt, from 10nt to 25 nt, from 15 nt to 45 nt, from 15 nt to 40 nt, from 15 nt to 35nt, from 15 nt to 30 nt, from 15 nt to 25, or from 17 nt to 23 nt).

As will be described in greater detail below, aspects of the methods caninclude fixation, permeabilization, dehydration, rehydration,post-fixation, nuclease inhibition, sample storage, target detection,signal amplification, and signal detection. In some instances, themethods include contacting a cellular sample with a fixation reagent anda permeabilization reagent to produce a fixed/permeabilized cellularsample; contacting the fixed/permeabilized cellular sample with anaqueous post-fixation reagent to produce a rehydrated/fixed cellularsample; contacting the rehydrated/fixed cellular sample with a nucleaseinhibitor; and evaluating the nuclease-contacted rehydrated/fixedcellular sample for the presence of the target nucleic acid (e.g.,ribonucleic acid, deoxyribonucleic acid).

Fixation

Aspects of the invention include “fixing” a cellular sample. The term“fixing” or “fixation” as used herein is the process of preservingbiological material (e.g., tissues, cells, organelles, molecules, etc.)from decay and/or degradation. Fixation may be accomplished using anyconvenient protocol. Fixation can include contacting the cellular samplewith a fixation reagent (i.e., a reagent that contains at least onefixative). Cellular samples can be contacted by a fixation reagent for awide range of times, which can depend on the temperature, the nature ofthe sample, and on the fixative(s). For example, a cellular sample canbe contacted by a fixation reagent for 24 or less hours, 18 or lesshours, 12 or less hours, 8 or less hours, 6 or less hours, 4 or lesshours, 2 or less hours, 60 or less minutes, 45 or less minutes, 30 orless minutes, 25 or less minutes, 20 or less minutes, 15 or lessminutes, 10 or less minutes, 5 or less minutes, or 2 or less minutes.

A cellular sample can be contacted by a fixation reagent for a period oftime in a range of from 5 minutes to 24 hours (e.g., from 10 minutes to20 hours, from 10 minutes to 18 hours, from 10 minutes to 12 hours, from10 minutes to 8 hours, from 10 minutes to 6 hours, from 10 minutes to 4hours, from 10 minutes to 2 hours, from 15 minutes to 20 hours, from 15minutes to 18 hours, from 15 minutes to 12 hours, from 15 minutes to 8hours, from 15 minutes to 6 hours, from 15 minutes to 4 hours, from 15minutes to 2 hours, from 15 minutes to 1.5 hours, from 15 minutes to 1hour, from 10 minutes to 30 minutes, from 15 minutes to 30 minutes, from30 minutes to 2 hours, from 45 minutes to 1.5 hours, or from 55 minutesto 70 minutes).

A cellular sample can be contacted by a fixation reagent at varioustemperatures, depending on the protocol and the reagent used. Forexample, in some instances a cellular sample can be contacted by afixation reagent at a temperature ranging from −22° C. to 55° C., wherespecific ranges of interest include, but are not limited to: 50 to 54°C., 40 to 44° C., 35 to 39° C., 28 to 32° C., 20 to 26° C., 0 to 6° C.,and −18 to −22° C. In some instances a cellular sample can be contactedby a fixation reagent at a temperature of −20° C., 4° C., roomtemperature (22-25° C.), 30° C., 37° C., 42° C., or 52° C.

Any convenient fixation reagent can be used. Common fixation reagentsinclude crosslinking fixatives, precipitating fixatives, oxidizingfixatives, mercurials, and the like. Crosslinking fixatives chemicallyjoin two or more molecules by a covalent bond and a wide range ofcross-linking reagents can be used. Examples of suitable cross-likingfixatives include but are not limited to aldehydes (e.g., formaldehyde,also commonly referred to as “paraformaldehyde” and “formalin”;glutaraldehyde; etc.), imidoesters, NHS (N-Hydroxysuccinimide) esters,and the like. Examples of suitable precipitating fixatives include butare not limited to alcohols (e.g., methanol, ethanol, etc.), acetone,acetic acid, etc. In some embodiments, the fixative is formaldehyde(i.e., paraformaldehyde or formalin). A suitable final concentration offormaldehyde in a fixation reagent is 0.1 to 10%, 1-8%, 1-4%, 1-2%,3-5%, or 3.5-4.5%. In some embodiments the cellular sample is fixed in afinal concentration of 4% formaldehyde (as diluted from a moreconcentrated stock solution, e.g., 38%, 37%, 36%, 20%, 18%, 16%, 14%,10%, 8%, 6%, etc.). In some embodiments the cellular sample is fixed ina final concentration of 10% formaldehyde. In some embodiments thecellular sample is fixed in a final concentration of 1% formaldehyde. Insome embodiments, the fixative is glutaraldehyde. A suitableconcentration of glutaraldehyde in a fixation reagent is 0.1 to 1%.

A fixation reagent can contain more than one fixative in anycombination. For example, in some embodiments the cellular sample iscontacted with a fixation reagent containing both formaldehyde andglutaraldehyde.

Permeabilization

Aspects of the invention include “permeabilizing” a cellular sample. Theterms “permeabilization” or “permeabilize” as used herein refer to theprocess of rendering the cells (cell membranes etc.) of a cellularsample permeable to experimental reagents such as nucleic acid probes,antibodies, chemical substrates, etc. Any convenient method and/orreagent for permeabilization can be used. Suitable permeabilizationreagents include detergents (e.g., Saponin, Triton X-100, Tween-20,etc.), organic fixatives (e.g., acetone, methanol, ethanol, etc.),enzymes, etc. Detergents can be used at a range of concentrations. Forexample, 0.001%-1% detergent, 0.05%-0.5% detergent, or 0.1%-0.3%detergent can be used for permeabilization (e.g., 0.1% Saponin, 0.2%tween-20, 0.1-0.3% triton X-100, etc.).

In some embodiments, the same solution can be used as the fixationreagent and the permeabilization reagent. For example, in someembodiments, the fixation reagent contains 0.1%-10% formaldehyde and0.001%-1% saponin. In some embodiments, the fixation reagent contains 1%formaldehyde and 0.3% saponin.

A cellular sample can be contacted by a permeabilization reagent for awide range of times, which can depend on the temperature, the nature ofthe sample, and on the permeabilization reagent(s). For example, acellular sample can be contacted by a permeabilization reagent for 24 ormore hours (see storage described below), 24 or less hours, 18 or lesshours, 12 or less hours, 8 or less hours, 6 or less hours, 4 or lesshours, 2 or less hours, 60 or less minutes, 45 or less minutes, 30 orless minutes, 25 or less minutes, 20 or less minutes, 15 or lessminutes, 10 or less minutes, 5 or less minutes, or 2 or less minutes. Acellular sample can be contacted by a permeabilization reagent atvarious temperatures, depending on the protocol and the reagent used.For example, in some instances a cellular sample can be contacted by apermeabilization reagent at a temperature ranging from −82° C. to 55°C., where specific ranges of interest include, but are not limited to:50 to 54° C., 40 to 44° C., 35 to 39° C., 28 to 32° C., 20 to 26° C., 0to 6° C., −18 to −22° C., and −78 to −82° C. In some instances acellular sample can be contacted by a permeabilization reagent at atemperature of −80° C., −20° C., 4° C., room temperature (22-25° C.),30° C., 37° C., 42° C., or 52° C.

In some embodiments, a cellular sample is contacted with an enzymaticpermeabilization reagent. Enzymatic permeabilization reagents thatpermeabilize a cellular sample by partially degrading extracellularmatrix or surface proteins that hinder the permeation of the cellularsample by assay reagents. Contact with an enzymatic permeabilizationreagent can take place at any point after fixation and prior to targetdetection. In some instances the enzymatic permeabilization reagent isproteinase K, a commercially available enzyme. In such cases, thecellular sample is contacted with proteinase K prior to contact with apost-fixation reagent (described below). Proteinase K treatment (i.e.,contact by proteinase K; also commonly referred to as “proteinase Kdigestion”) can be performed over a range of times at a range oftemperatures, over a range of enzyme concentrations that are empiricallydetermined for each cell type or tissue type under investigation. Forexamples, a cellular sample can be contacted by proteinase K for 30 orless minutes, 25 or less minutes, 20 or less minutes, 15 or lessminutes, 10 or less minutes, 5 or less minutes, or 2 or less minutes. Acellular sample can be contacted by 1 ug/ml or less, 2 ug/m or less l, 4ug/ml or less, 8 ug/ml or less, 10 ug/ml or less, 20 ug/ml or less, 30ug/ml or less, 50 ug/ml or less, or 100 ug/ml or less proteinase K. Acellular sample can be contacted by proteinase K at a temperatureranging from 2° C. to 55° C., where specific ranges of interest include,but are not limited to: 50 to 54° C., 40 to 44° C., 35 to 39° C., 28 to32° C., 20 to 26° C., and 0 to 6° C. In some instances a cellular samplecan be contacted by proteinase K at a temperature of 4° C., roomtemperature (22-25° C.), 30° C., 37° C., 42° C., or 52° C. In someembodiments, a cellular sample is not contacted with an enzymaticpermeabilization reagent. In some embodiments, a cellular sample is notcontacted with proteinase K.

Contact of a cellular sample with at least a fixation reagent and apermeabilization reagent results in the production of afixed/permeabilized cellular sample.

Dehydration

Aspects of the invention include “dehydrating” a cellular sample. Theterm “dehydration” as used herein refers to the replacement of anaqueous sample diluent with a non-aqueous diluent (i.e., a dehydrationreagent, e.g., an alcohol). In some embodiments, dehydration refers tothe replacement of aqueous solution from a cellular sample with analcohol (e.g., methanol, ethanol).

A cellular sample can be contacted by a dehydration reagent for 12months or less, 6 months or less, 2 months or less, 2 weeks or less, 24hours or less, 18 hours or less, 12 hours or less, 8 hours or less, 6hours or less, 4 hours or less, 2 hours or less, 60 minutes or less, 45minutes or less, 30 minutes or less, 25 minutes or less, 20 minutes orless, 15 minutes or less, 10 minutes or less, 5 minutes or less, or 2minutes or less. A cellular sample can be contacted by a dehydrationreagent at various temperatures, depending on the protocol. For example,in some instances a cellular sample can be contacted by a dehydrationreagent at a temperature ranging from −82° C. to 55° C., where specificranges of interest include, but are not limited to: 50 to 54° C., 40 to44° C., 35 to 39° C., 28 to 32° C., 20 to 26° C., 0 to 6° C., −18 to−22° C., and −78 to −82° C. In some instances a cellular sample can becontacted by a dehydration reagent at a temperature of −80° C., −20° C.,4° C., room temperature (22-25° C.), 30° C., 37° C., 42° C., or 52° C.

In cases where an alcohol (e.g., methanol or ethanol) is used as apermeabilization reagent (see previous section), permeabilization anddehydration occur simultaneously and the step can simply be referred toas a permeabilization step. As such, when an alcohol is used as apermeabilization reagent, the cellular sample is a dehydratedfixed/permeabilized cellular sample. Therefore, the term“fixed/permeabilized cellular sample” can be used to describe either acellular sample that has not been dehydrated (e.g., when thepermeabilization reagent is not an alcohol) or a cellular sample thathas been dehydrated (e.g., when the permeabilization reagent is analcohol). As described below, a cellular sample that has been dehydratedcan be stored prior to rehydration.

Rehydration

Aspects of the invention include “rehydrating” a cellular sample. Theterm “rehydration” as used herein refers to the replacement of anon-aqueous solution such as alcohol (e.g., methanol, ethanol, etc.)with an aqueous solution (i.e., a rehydration reagent). The replacementcan be accomplished by centrifugation to pellet the cells of thecellular sample, removing the supernatant, and replacing the alcoholwith a new solution. Rehydration can be performed in one step byreplacing the alcohol with a fully aqueous solution (e.g., a solutionthat does not contain alcohol) or it can be performed in more than onestep by re-suspending the cells in a series of solutions with decreasingalcohol content. For example, a two-step rehydration can include thereplacement of 100% methanol with 50% methanol, followed by replacementwith a fully aqueous solution; and a four-step rehydration can includethe replacement of 100% methanol with 75% methanol, followed byreplacement with 50% methanol, followed by replacement with 25%methanol, followed by replacement with a fully aqueous solution.

A cellular sample can be contacted by a rehydration reagent for 60minutes or less, 45 minutes or less, 30 minutes or less, 25 minutes orless, 20 minutes or less, 15 minutes or less, 10 minutes or less, 5minutes or less, or 2 minutes or less. A cellular sample can becontacted by a rehydration reagent at various temperatures, depending onthe protocol. For example, a cellular sample can be contacted by arehydration reagent at a temperature ranging from 2° C. to 55° C., wherespecific ranges of interest include, but are not limited to: 50 to 54°C., 40 to 44° C., 35 to 39° C., 28 to 32° C., 20 to 26° C., and 0 to 6°C. In some instances a cellular sample can be contacted by a rehydrationreagent at a temperature of 4° C., room temperature (22-25° C.), 30° C.,37° C., 42° C., or 52° C.

Once rehydrated, the sample can be referred to as a rehydrated cellularsample. Contact of a cellular sample with at least a fixation reagent, adehydration reagent, and a rehydration reagent results in the productionof a rehydrated/fixed cellular sample. The term “rehydrated/fixedcellular sample” can therefore be used to describe either a cellularsample that has been post-fixed (see below, e.g., when the rehydrationreagent includes a fixative) or a cellular sample that has not beenpost-fixed (e.g., when the rehydration reagent does not include afixative).

Post-Fixation

Aspects of the invention include “post-fixing” a cellular sample Theterm “post-fixation” as used herein refers to the process ofre-contacting a cellular sample with at least one post-fixation reagent(i.e., a reagent containing at least one fixative). This step can alsobe considered a “secondary fixation” step because the cellular samplehas previously been contacted by a fixation reagent. Suitablepost-fixation reagents are the same as the suitable fixation reagentsand the cellular sample can be contacted by a post-fixation reagent atthe same concentrations (e.g., same range of concentrations), for thesame periods of time (e.g., range of times), at the same temperatures(e.g., ranges of temperatures) as for the fixation step. Thepost-fixation protocol (i.e., the reagent used, the contact time, thetemperature, etc) can be the same as that used for the fixation step orit can be different than the fixation step. For example, the cellularsample can be contacted by a post-fixation reagent that is the same asor different than the fixation reagent which it previously contacted.

In some embodiments the cellular sample is post-fixed (i.e., contactedby a post-fixation reagent, e.g., an aqueous post-fixation reagent)after a rehydration step. In some embodiments, the aqueous solution(i.e., rehydration reagent) used to replace the alcohol duringrehydration is also a fixation reagent (i.e., an aqueous post-fixationreagent that contains a fixative, e.g., 1% formaldehyde, 4%formaldehyde, etc.) and the rehydration step is therefore also apost-fixation step.

Nuclease Inhibition

Aspects of the invention include contacting a cellular sample with anuclease inhibitor. As used herein, a “nuclease inhibitor” is anymolecule that can be used to inhibit nuclease activity within thecellular sample such that integrity of the nucleic acids within thecells of the cellular sample is preserved. In other words, degradationof the nucleic acids within the cells of the cellular sample by nucleaseactivity is inhibited by contacting the cellular sample with a nucleaseinhibitor. In some embodiments, the nuclease inhibitor is an RNaseinhibitor (i.e., the inhibitor inhibits RNase activity). Examples ofsuitable commercially available nuclease inhibitors include, but are notlimited to non-protein based inhibitors (e.g., aurintricarboxylic acid(ATA); Diethyl Pyrocarbonate (DEPC); RNAsecure™ Reagent from LifeTechnologies; and the like) and protein based inhibitors (e.g.,ribonuclease inhibitor from EMD Millipore; RNaseOUT™ RecombinantRibonuclease Inhibitor, SUPERaseIn™, ANTI-RNase, and RNase Inhibitorfrom Life Technologies; RNase Inhibitor and Protector RNase Inhibitorfrom Roche; RNAsin from Promega, and the like). Nuclease inhibitors canbe used at a range of concentrations as recommended by their commercialsources.

A cellular sample can be contacted by a nuclease inhibitor at any timeprior to the target nucleic acid detection step (see below). Forexample, a cellular sample can be contacted with a nuclease inhibitorafter the sample is rehydrated and post-fixed to produce a nucleaseinhibitor-contacted rehydrated/fixed cellular sample.

Storage

A cellular sample can be stored, when desired, for an extended period oftime prior to the target nucleic acid detection step. In some instances,the extended period of time is 1 week or longer, e.g., 1 month orlonger, including 6 months or longer, e.g., 1 year or longer. In someinstances, the extended period of time ranges from 1 week to 5 years,e.g., 2 weeks to 4 years, 4 weeks to 3 years, 8 weeks to 2 years, or 12weeks to 1 year. In some instances, the cellular sample can be stored ata temperature below 0° C. (e.g., below −19° C., below −79° C., etc.) asa dehydrated cellular sample (i.e., while being contacted by adehydration reagent and prior to rehydration). In some embodiments, thesample can be stored as a nuclease inhibitor-contacted rehydrated/fixedcellular sample, prior to target nucleic acid detection. In such cases,the cellular sample is stored at 2-6° C. in an aqueous buffer thatincludes a nuclease inhibitor.

Protein Detection Reagents

Aspects of the invention may include contacting the cellular sample witha protein detection reagent. The term “protein detection reagent” asused herein refers to any reagent that specifically binds to a targetprotein (e.g., a target protein of a cell of the cellular sample) andfacilitates the qualitative and/or quantitative detection of the targetprotein. The terms “specific binding,” “specifically binds,” and thelike, refer to the preferential binding to a molecule relative to othermolecules or moieties in a solution or reaction mixture. In someembodiments, the affinity between protein detection reagent and thetarget protein to which it specifically binds when they are specificallybound to each other in a binding complex is characterized by a K_(d)(dissociation constant) of 10⁻⁶ M or less, such as 10⁻⁷ M or less,including 10⁻⁸ M or less, e.g., 10⁻⁹ M or less, 10⁻¹⁰ M or less, 10⁻¹¹ Mor less, 10⁻¹² M or less, 10⁻¹³ M or less, 10⁻¹⁴ M or less, including10⁻¹⁵ M or less. “Affinity” refers to the strength of binding, increasedbinding affinity being correlated with a lower K_(d).

In some embodiments, a protein detection reagent includes a label or alabeled binding member. A “label” or “label moiety” is any moiety thatprovides for signal detection and may vary widely depending on theparticular nature of the assay. Label moieties of interest include bothdirectly and indirectly detectable labels. Suitable labels for use inthe methods described herein include any moiety that is indirectly ordirectly detectable by spectroscopic, photochemical, biochemical,immunochemical, electrical, optical, chemical, or other means. Forexample, suitable labels include biotin for staining with labeledstreptavidin conjugate, a fluorescent dye (e.g., fluorescein, Texas red,rhodamine, a flurochrome label such as an ALEXA FLUOR® label, and thelike), a radiolabel (e.g., ³H, ¹²⁵I, ³⁵S, ¹⁴C, or ³²P), an enzyme (e.g.,peroxidase, alkaline phosphatase, galactosidase, and others commonlyused in an ELISA), a fluorescent protein (e.g., green fluorescentprotein, red fluorescent protein, yellow fluorescent protein, and thelike), a metal label, a colorimetric label, and the like. Fluorescentlabels can be detected using a photodetector (e.g., in a flow cytometer)to detect emitted light. Enzymatic labels are typically detected byproviding the enzyme with a substrate and detecting the reaction productproduced by the action of the enzyme on the substrate, and colorimetriclabels are detected by simply visualizing the colored label.

Metal labels (e.g., Sm¹⁵², Tb¹⁵⁹, Er¹⁷⁰, Nd¹⁴⁶, Nd¹⁴², and the like) canbe detected (e.g., the amount of label can be measured) using anyconvenient method, including, for example, mass cytometry (see, forexample: (i) U.S. Pat. No. 7,479,630; (ii) Wang et al., Cytometry A.2012 July; 81(7):567-75: “Human CD4+ lymphocytes for antigenquantification: characterization using conventional flow cytometry andmass cytometry”; (iii) Bandura et. al., Anal Chem. 2009 Aug. 15;81(16):6813-22: “Mass cytometry: technique for real time single cellmultitarget immunoassay based on inductively coupled plasmatime-of-flight mass spectrometry”; and (iv) Ornatsky et. al., J ImmunolMethods. 2010 Sep. 30; 361(1-2):1-20: “Highly multiparametric analysisby mass cytometry”; all of which are hereby incorporated by reference intheir entirety), which, as with flow cytometry, is a method of singlecell analysis. As described above, mass cytometry is a real-timequantitative analytical technique whereby cells or particles areindividually introduced into a mass spectrometer (e.g., InductivelyCoupled Plasma Mass Spectrometer (ICP-MS)), and a resultant ion cloud(or multiple resultant ion clouds) produced by a single cell is analyzed(e.g., multiple times) by mass spectrometry (e.g., time of-flight massspectrometry). Mass cytometry can use elements (e.g., a metal) or stableisotopes, attached as label moieties to a detection reagent (e.g., anantibody and/or a nucleic acid detection agent).

In some instances, a protein detection reagent is a polyclonal ormonoclonal antibody or a binding fragment thereof (i.e., an antibodyfragment that is sufficient to bind to the target of interest, e.g., theprotein target). Antibody fragments (i.e., binding fragments) can be,for example, monomeric Fab fragments, monomeric Fab′ fragments, ordimeric F(ab)′₂ fragments. Also within the scope of the term “antibodyor a binding fragment thereof” are molecules produced by antibodyengineering, such as single-chain antibody molecules (scFv) or humanizedor chimeric antibodies produced from monoclonal antibodies byreplacement of the constant regions of the heavy and light chains toproduce chimeric antibodies or replacement of both the constant regionsand the framework portions of the variable regions to produce humanizedantibodies.

Stimulating Agents

Aspects of the invention may include contacting the cellular sample witha “stimulating agent”, also referred to herein as a “stimulator.” Bystimulating agent it is meant any compound that affects at least onecellular activity or that alters the cellular steady state (i.e.,induced or reduced in abundance or activity). Contacting a cellularsample with a stimulating agent can be used to ascertain the cellularresponse to the agent. By “effective amount” of a stimulating agent, itis meant that a stimulating agent is present in an amount to affect atleast one cellular activity that alters the cellular steady state (i.e.,induced or reduced in abundance or activity). A stimulating agent can beprovided as a powder or as a liquid. As such, a stimulating agent caninclude various compounds and formulations, such as intracellular signalinducing and immunomodulatory agents. Examples include small moleculedrugs as well as peptides, proteins, lipids carbohydrates and the like.Of particular interest are compounds such as type I interferons (e.g.,IFN-α, IFN-β), interleukins (e.g., interleukin-2 (IL-2), IL-4, IL-6,IL-7, IL-10, IL-12, IL-15, IL-21), tumor necrosis factor alpha (TNF-α),gamma interferon (IFN-γ), transforming growth factor β, and the like. Insome embodiments, the stimulating agent includes an immunomodulatorycytokine, such as immunomodulatory cytokines represented by interferons,interleukins, and chemokines among others. Interferon alpha is ofspecific interest.

Target Nucleic Acid Detection and Signal Amplification

The subject methods are methods of assaying for the presence of a targetnucleic acid. As such, the subject methods are methods (when a targetnucleic acid is present in a cell of a cellular sample) of detecting thetarget nucleic acid, producing a signal in response to target nucleicacid detection, and detecting the produced signal. The signal producedby a detected target nucleic acid can be any detectable signal (e.g., afluorescent signal, an amplified fluorescent signal, a chemiluminescentsignal, etc.)

Aspects of the invention include methods of detecting a target nucleicacid (i.e., target nucleic acid detection). In some embodiments, thecellular sample is contacted with a nucleic acid detection agent. Asused herein, the term “nucleic acid detection agent” means any reagentthat can specifically bind to a target nucleic acid. For example,suitable nucleic acid detection agents can be nucleic acids (or modifiednucleic acids) that are at least partially complementary to andhybridize with a sequence of the target nucleic acid. In someembodiments, the nucleic acid detection agent includes a probe or set ofprobes (i.e., probe set), each of which specifically binds (i.e.,hybridizes to) a sequence (i.e., target sequence) of the target nucleicacid.

A nucleic acid is “complementary” or “hybridizable” to another nucleicacid, such as a cDNA, genomic DNA, or RNA, when a single stranded formof the nucleic acid can “base-pair” with or “anneal” to the othernucleic acid in a sequence specific manner (i.e., a nucleic acidspecifically binds to a complementary nucleic acid) under theappropriate in vitro or in vivo conditions of temperature and solutionionic strength. Hybridization requires that the two nucleic acidscontain complementary sequences although mismatches between bases arepossible such that 100% complementarity is not an absolute requirement.The conditions appropriate for hybridization between two nucleic acidsdepend on the length of the nucleic acids and the degree ofcomplementarity, where the greater the degree of complementarity, thestronger the hybridization (i.e., the greater the value of the meltingtemperature (Tm)). In some instances, suitable hybridizable nucleicacids are composed of 10 or more, 15 or more, 20 or more, 25 or more, or30 or more nucleotides. The skilled artisan will recognize that thestrength (i.e., degree) of hybridization also depends on temperature andwash solution salt concentration, both of which may be adjusted asnecessary. Percent complementarity between particular stretches ofnucleic acid sequences within nucleic acids can be determined routinelyusing readily available sequence alignment programs (e.g., BLAST (basiclocal alignment search tools)).

In some embodiments, a nucleic acid detection agent includes a signalproducing system. A signal producing system can have one or morecomponents and can be any system that provides a signal when the nucleicacid detection agent detects a target nucleic acid. In some instances, asignal producing system includes a labeled nucleic acid probe. A labelednucleic acid probe is a nucleic acid that is labeled with any labelmoiety. As such, a nucleic acid detection agent can include a signalproducing system that includes a labeled nucleic acid probe. In someembodiments, the nucleic acid detection agent is a single labeledmolecule (i.e., a labeled nucleic acid probe) that specifically binds tothe target nucleic acid. In some embodiments, the nucleic acid detectionagent includes multiple molecules, one of which specifically binds tothe target nucleic acid. In such embodiments, when a labeled nucleicacid probe is present, the labeled nucleic acid probe does notspecifically bind to the target nucleic acid, but instead specificallybinds to one of the other molecules of the nucleic acid detection agent.

A “label” or “label moiety” for a nucleic acid probe is any moiety thatprovides for signal detection and may vary widely depending on theparticular nature of the assay. Label moieties of interest include bothdirectly and indirectly detectable labels. Suitable labels for use inthe methods described herein include any moiety that is indirectly ordirectly detectable by spectroscopic, photochemical, biochemical,immunochemical, electrical, optical, chemical, or other means. Forexample, suitable labels include antigenic labels (e.g., digoxigenin(DIG), fluorescein, dinitrophenol (DNP), etc.), biotin for staining withlabeled streptavidin conjugate, a fluorescent dye (e.g., fluorescein,Texas red, rhodamine, a flurochrome label such as an ALEXA FLUOR® label,and the like), a radiolabel (e.g., ³H, ¹²⁵I, ³⁵S, ¹⁴C, or ³²P), anenzyme (e.g., peroxidase, alkaline phosphatase, galactosidase, andothers commonly used in an ELISA), a fluorescent protein (e.g., greenfluorescent protein, red fluorescent protein, yellow fluorescentprotein, and the like), a metal label, a colorimetric label, and thelike. An antigenic label can be incorporated into the nucleic acid onany nucleotide (e.g., A,U,G,C).

Fluorescent labels can be detected using a photodetector (e.g., in aflow cytometer) to detect emitted light. Enzymatic labels are typicallydetected by providing the enzyme with a substrate and detecting thereaction product produced by the action of the enzyme on the substrate,colorimetric labels can be detected by simply visualizing the coloredlabel, and antigenic labels can be detected by providing an antibody (ora binding fragment thereof) that specifically binds to the antigeniclabel. An antibody that specifically binds to an antigenic label can bedirectly or indirectly detectable. For example, the antibody can beconjugated to a label moiety (e.g., a fluorophore) that provides thesignal (e.g., fluorescence); the antibody can be conjugated to an enzyme(e.g., peroxidase, alkaline phosphatase, etc.) that produces adetectable product (e.g., fluorescent product) when provided with anappropriate substrate (e.g., fluorescent-tyramide, FastRed, etc.); etc.

Metal labels (e.g., Sm¹⁵², Tb¹⁵⁹, Er¹⁷⁰, Nd¹⁴⁶, Nd¹⁴², and the like) canbe detected (e.g., the amount of label can be measured) using anyconvenient method, including, for example, mass cytometry (see, forexample: (i) U.S. Pat. No. 7,479,630; (ii) Wang et al., Cytometry A.2012 July; 81(7):567-75: “Human CD4+ lymphocytes for antigenquantification: characterization using conventional flow cytometry andmass cytometry”; (iii) Bandura et. al., Anal Chem. 2009 Aug. 15;81(16):6813-22: “Mass cytometry: technique for real time single cellmultitarget immunoassay based on inductively coupled plasmatime-of-flight mass spectrometry”; and (iv) Ornatsky et. al., J ImmunolMethods. 2010 Sep. 30; 361(1-2):1-20: “Highly multiparametric analysisby mass cytometry”), which, as with flow cytometry, is a method ofsingle cell analysis. As described above, mass cytometry is a real-timequantitative analytical technique whereby cells or particles areindividually introduced into a mass spectrometer (e.g., InductivelyCoupled Plasma Mass Spectrometer (ICP-MS)), and a resultant ion cloud(or multiple resultant ion clouds) produced by a single cell is analyzed(e.g., multiple times) by mass spectrometry (e.g., time of-flight massspectrometry). Mass cytometry can use elements (e.g., a metal) or stableisotopes, attached as label moieties to a detection reagent (e.g., anantibody and/or a nucleic acid detection agent).

In some cases the signal produced by the detection of the target nucleicacid is amplified prior to signal detection. As such, a signal producingsystem of a nucleic acid detection agent can include a signalamplification component. A signal amplification component is anycomponent that facilitates the amplification of signal. Examples ofsignal amplification components include but are not limited to enzymes(either unconjugated or conjugated to an antibody for fragment thereof)that produce detectable products when contacted with appropriatesubstrate; branched nucleic acids; and the like. In some instances, asignal amplification component is a branched nucleic acid. In suchcases, the signal amplification can be referred to as branched nucleicacid (e.g., DNA) signal amplification (e.g., bDNA signal amplification).A branched nucleic acid (e.g., bDNA) is a series of nucleic acids(and/or modified nucleic acids, as described below) that hybridize toeach other (e.g., in multiple locations), thus taking on a branch-shapedstructure.

In some bDNA assays for gene expression analysis, a target nucleic acid(e.g., an RNA, e.g., an mRNA, a microRNA, etc.) whose expression is tobe detected is released from cells and captured by a Capture Probe (CP)on a solid surface (e.g., a well of a microtiter plate) throughsynthetic oligonucleotide probes called Capture Extenders (CEs). Eachcapture extender has a first polynucleotide sequence that can hybridizeto the target nucleic acid and a second polynucleotide sequence that canhybridize to the capture probe. In some cases, two or more captureextenders are used. Probes of another type, called Label Extenders(LEs), hybridize to different sequences on the target nucleic acid andto sequences on an amplification multimer. Additionally, Blocking Probes(BPs), which hybridize to regions of the target nucleic acid notoccupied by CEs or LEs, can be used to reduce non-specific target probebinding. A probe set for a given nucleic acid thus consists of CEs, LEs,and/or BPs for the target nucleic acid. The CEs, LEs, and BPs arecomplementary to nonoverlapping sequences in the target nucleic acid,and can be contiguous.

Signal amplification begins with the binding of the LEs to the targetnucleic acid. An amplification multimer can then be hybridized to theLEs. The amplification multimer can have multiple copies of a sequencethat is complementary to a label probe (the amplification multimer canbe a branched-chain nucleic acid; for example, the amplificationmultimer can be a branched, forked, or comb-like nucleic acid or alinear nucleic acid). A label can be covalently attached to each labelprobe, as discussed above for a nucleic probe (e.g., a fluorescentlabel, e.g., a fluorescent dye, a fluorochrome label, etc.; an enzyme,e.g., peroxidase, alkaline phosphatase, galactosidase, etc.; a metallabel; and the like). Alternatively, the label can be noncovalentlybound to the label probes. Labeled complexes can then be detected (e.g.,by fluorescence detection; by the alkaline phosphatase-mediateddegradation of a chemilumigenic substrate, e.g., dioxetane; etc.).Luminescence and/or fluorescence can be reported in any convenient way(e.g., as relative fluorescence units, as relative light unit (RLUs) ona microplate reader, etc.). The amount of chemiluminescence isproportional to the level of target nucleic acid.

In the preceding example, the amplification multimer and the labelprobes comprise a label probe system. In another example, the labelprobe system also comprises a preamplifier, e.g., as described in U.S.Pat. Nos. 5,635,352 and 5,681,697, which further amplifies the signalfrom a single target nucleic acid. In yet another example, the labelextenders hybridize directly to the label probes and no amplificationmultimer or preamplifier is used, so the signal from a single targetnucleic acid molecule is only amplified by the number of distinct labelextenders that hybridize to the target nucleic acid.

Basic bDNA assays have been well described. See, e.g., U.S. Pat. No.4,868,105 to Urdea et al. entitled “Solution phase nucleic acid sandwichassay”; U.S. Pat. No. 5,635,352 to Urdea et al. entitled “Solution phasenucleic acid sandwich assays having reduced background noise”; U.S. Pat.No. 5,681,697 to Urdea et al. entitled “Solution phase nucleic acidsandwich assays having reduced background noise and kits therefor”; U.S.Pat. No. 5,124,246 to Urdea et al. entitled “Nucleic acid multimers andamplified nucleic acid hybridization assays using same”; U.S. Pat. No.5,624,802 to Urdea et al. entitled “Nucleic acid multimers and amplifiednucleic acid hybridization assays using same”; U.S. Pat. No. 5,849,481to Urdea et al. entitled “Nucleic acid hybridization assays employinglarge comb-type branched polynucleotides”; U.S. Pat. No. 5,710,264 toUrdea et al. entitled “Large comb type branched polynucleotides”; U.S.Pat. No. 5,594,118 to Urdea and Horn entitled “Modified N-4 nucleotidesfor use in amplified nucleic acid hybridization assays”; U.S. Pat. No.5,093,232 to Urdea and Horn entitled “Nucleic acid probes”; U.S. Pat.No. 4,910,300 to Urdea and Horn entitled “Method for making nucleic acidprobes”; U.S. Pat. Nos. 5,359,100; 5,571,670; 5,614,362; 6,235,465;5,712,383; 5,747,244; 6,232,462; 5,681,702; 5,780,610; 5,780,227 toSheridan et al. entitled “Oligonucleotide probe conjugated to a purifiedhydrophilic alkaline phosphatase and uses thereof”; U.S. patentapplication Publication No. US2002172950 by Kenny et al. entitled“Highly sensitive gene detection and localization using in situbranched-DNA hybridization”; Wang et al. (1997) “Regulation of insulinpreRNA splicing by glucose” Proc Nat Acad Sci USA 94:4360-4365; Collinset al. (1998) “Branched DNA (bDNA) technology for direct quantificationof nucleic acids: Design and performance” in Gene Quantification, FFerre, ed.; and Wilber and Urdea (1998) “Quantification of HCV RNA inclinical specimens by branched DNA (bDNA) technology” Methods inMolecular Medicine: Hepatitis C 19:71-78.

Additional exemplary publications that describe branched nucleic acidsand methods that use bDNA assays (including multiplex assays) alsoinclude, for example, U.S. patent applications: 2006/0263769,2009/0081688, 2010/0099175, 2012/0003648, 2012/0004132, 2012/0052498,2012/0172246, 2012/0071343, 2012/0214152, 2012/0100540, 2013/0023433,and 2013/0171621; as well as U.S. Pat. Nos. 7,033,758; 7,709,198;7,803,541; and 8,114,681, all of which are hereby incorporated byreference in their entirety.

FIG. 1A schematically depicts an embodiment of the invention thatincludes a branched nucleic acid signal amplification component 3 and 4to detect a target nucleic acid 1. The nucleic acid detection agent 2-5of FIG. 1A includes a probe set 2 and a signal producing system 3-5having both a labeled nucleic acid probe 5 (i.e., detector) and abranched nucleic acid 3 (i.e., pre-amplifier) and 4 (i.e., amplifier).The steps and elements of the target detection and signal productiondepicted in FIG. 1A are described in U.S. applications US 2010/0099175and US 2009/0081688. In FIG. 1A the target nucleic acid 1 is detected bythe probe set 2, which is made up of multiple oligonucleotides (probes)that hybridize to various predetermined segments along the targetnucleic acid 1. Several molecules 3 (i.e., pre-amplifier) and 4 (i.e.,amplifier) of the branched nucleic acid hybridize to each other, thuscreating a branched nucleic acid structure. A labeled nucleic acid probe5 (i.e., detector) specifically binds to the branched nucleic acid 3 and4. Thus, each oligonucleotide of the probe set 2 will be marked byseveral labeled nucleic acid probes 5 via signal amplification by thebranched nucleic acid 3 and 4.

Modified Nucleic Acids

In some embodiments, a nucleic acid detection agent (e.g., a probe) is amodified nucleic acid. A modified nucleic acid has one or moremodifications, e.g., a base modification, a backbone modification, etc,to provide the nucleic acid with a new or enhanced feature (e.g.,improved stability). A nucleoside can be a base-sugar combination, thebase portion of which is a heterocyclic base. Heterocyclic bases includethe purines and the pyrimidines. Nucleotides are nucleosides thatfurther include a phosphate group covalently linked to the sugar portionof the nucleoside. For those nucleosides that include a pentofuranosylsugar, the phosphate group can be linked to the 2′, the 3′, or the 5′hydroxyl moiety of the sugar. In forming oligonucleotides, the phosphategroups covalently link adjacent nucleosides to one another to form alinear polymeric compound. In some cases, the respective ends of thislinear polymeric compound can be further joined to form a circularcompound. In addition, linear compounds may have internal nucleotidebase complementarity and may therefore fold in a manner as to produce afully or partially double-stranded compound. Within oligonucleotides,the phosphate groups can be referred to as forming the internucleosidebackbone of the oligonucleotide. The linkage or backbone of RNA and DNAcan be a 3′ to 5′ phosphodiester linkage.

—Modified Backbones and Modified Internucleoside Linkages—

Examples of suitable nucleic acids containing modifications includenucleic acids with modified backbones or non-natural internucleosidelinkages. Nucleic acids having modified backbones include those thatretain a phosphorus atom in the backbone and those that do not have aphosphorus atom in the backbone. Suitable modified oligonucleotidebackbones containing a phosphorus atom therein include, for example,phosphorothioates, chiral phosphorothioates, phosphorodithioates,phosphotriesters, aminoalkylphosphotriesters, methyl and other alkylphosphonates including 3′-alkylene phosphonates, 5′-alkylenephosphonates and chiral phosphonates, phosphinates, phosphoramidatesincluding 3′-amino phosphoramidate and aminoalkylphosphoramidates,phosphorodiamidates, thionophosphoramidates, thionoalkylphosphonates,thionoalkylphosphotriesters, selenophosphates and boranophosphateshaving normal 3′-5′ linkages, 2′-5′ linked analogs of these, and thosehaving inverted polarity wherein one or more internucleotide linkages isa 3′ to 3′, 5′ to 5′ or 2′ to 2′ linkage. Suitable oligonucleotideshaving inverted polarity include a single 3′ to 3′ linkage at the3′-most internucleotide linkage i.e. a single inverted nucleosideresidue which may be a basic (the nucleobase is missing or has ahydroxyl group in place thereof). Various salts (such as, for example,potassium or sodium), mixed salts and free acid forms are also included.

In some embodiments, a subject nucleic acid has one or morephosphorothioate and/or heteroatom internucleoside linkages, inparticular —CH₂—NH—O—CH₂—, —CH₂—N(CH₃)—O—CH₂— (known as a methylene(methylimino) or MMI backbone), —CH₂—O—N(CH₃)—CH₂—,—CH₂—N(CH₃)—N(CH₃)—CH₂— and —O—N(CH₃)—CH₂—CH₂— (wherein the nativephosphodiester internucleotide linkage is represented as—O—P(═O)(OH)—O—CH₂—). MMI type internucleoside linkages are disclosed inthe above referenced U.S. Pat. No. 5,489,677. Suitable amideinternucleoside linkages are disclosed in t U.S. Pat. No. 5,602,240.

Also suitable are nucleic acids having morpholino backbone structures asdescribed in, e.g., U.S. Pat. No. 5,034,506. For example, in someembodiments, a subject nucleic acid includes a 6-membered morpholinoring in place of a ribose ring. In some of these embodiments, aphosphorodiamidate or other non-phosphodiester internucleoside linkagereplaces a phosphodiester linkage.

Suitable modified polynucleotide backbones that do not include aphosphorus atom therein have backbones that are formed by short chainalkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkylor cycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These includethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; riboacetyl backbones; alkene containingbackbones; sulfamate backbones; methyleneimino and methylenehydrazinobackbones; sulfonate and sulfonamide backbones; amide backbones; andothers having mixed N, O, S and CH₂ component parts.

—Mimetics—

A detection nucleic acid can be a nucleic acid mimetic. The term“mimetic” as it is applied to polynucleotides encompassespolynucleotides where only the furanose ring or both the furanose ringand the internucleotide linkage are replaced with non-furanose groups,replacement of only the furanose ring is also referred to as being asugar surrogate. The heterocyclic base moiety or a modified heterocyclicbase moiety is maintained for hybridization with an appropriate targetnucleic acid. One such nucleic acid, a polynucleotide mimetic that hasbeen shown to have excellent hybridization properties, is referred to asa peptide nucleic acid (PNA). In PNA, the sugar-backbone of apolynucleotide is replaced with an amide containing backbone, inparticular an aminoethylglycine backbone. The nucleotides are retainedand are bound directly or indirectly to aza nitrogen atoms of the amideportion of the backbone.

One polynucleotide mimetic that has excellent hybridization propertiesis a peptide nucleic acid (PNA). The backbone in PNA compounds is two ormore linked aminoethylglycine units which gives PNA an amide containingbackbone. The heterocyclic base moieties are bound directly orindirectly to aza nitrogen atoms of the amide portion of the backbone.Representative U.S. patents that describe the preparation of PNAcompounds include, but are not limited to: U.S. Pat. Nos. 5,539,082;5,714,331; and 5,719,262.

Another class of suitable polynucleotide mimetic is based on linkedmorpholino units (morpholino nucleic acid) having heterocyclic basesattached to the morpholino ring. A number of linking groups have beenreported that can link the morpholino monomeric units in a morpholinonucleic acid. One class of linking groups has been selected to give anon-ionic oligomeric compound. The non-ionic morpholino-based oligomericcompounds are less likely to have undesired interactions with cellularproteins. Morpholino-based polynucleotides are non-ionic mimics ofoligonucleotides which are less likely to form undesired interactionswith cellular proteins (Dwaine A. Braasch and David R. Corey,Biochemistry, 2002, 41(14), 4503-4510). Morpholino-based polynucleotidesare disclosed in U.S. Pat. No. 5,034,506. A variety of compounds withinthe morpholino class of polynucleotides have been prepared, having avariety of different linking groups joining the monomeric subunits.

Another suitable class of polynucleotide mimetic is referred to ascyclohexenyl nucleic acids (CeNA). The furanose ring normally present ina DNA/RNA molecule is replaced with a cyclohexenyl ring. CeNA DMTprotected phosphoramidite monomers have been prepared and used foroligomeric compound synthesis following classical phosphoramiditechemistry. Fully modified CeNA oligomeric compounds and oligonucleotideshaving specific positions modified with CeNA have been prepared andstudied (see Wang et al., J. Am. Chem. Soc., 2000, 122, 8595-8602). Theincorporation of CeNA monomers into a DNA chain increases the stabilityof a DNA/RNA hybrid. CeNA oligoadenylates formed complexes with RNA andDNA complements with similar stability to the native complexes. Theincorporation CeNA structures into natural nucleic acid structures wasshown by NMR and circular dichroism to proceed with conformationaladaptation.

Also suitable as modified nucleic acids are Locked Nucleic Acids (LNAs)and/or LNA analogs. In an LNA, the 2′-hydroxyl group is linked to the 4′carbon atom of the sugar ring thereby forming a 2′-C,4′-C-oxymethylenelinkage, and thereby forming a bicyclic sugar moiety. The linkage can bea methylene (—CH₂—), group bridging the 2′ oxygen atom and the 4′ carbonatom wherein n is 1 or 2 (Singh et al., Chem. Commun., 1998, 4,455-456). LNA and LNA analogs display very high duplex thermalstabilities with complementary DNA and RNA (Tm=+3 to +10° C.), stabilitytowards 3′-exonucleolytic degradation and good solubility properties.Potent and nontoxic oligonucleotides containing LNAs have been described(Wahlestedt et al., Proc. Natl. Acad. Sci. U.S.A., 2000, 97, 5633-5638).

The synthesis and preparation of the LNA monomers adenine, cytosine,guanine, 5-methyl-cytosine, thymine and uracil, along with theiroligomerization, and nucleic acid recognition properties have beendescribed (Koshkin et al., Tetrahedron, 1998, 54, 3607-3630). LNAs andpreparation thereof are also described in WO98/39352 and WO99/14226,both of which are hereby incorporated by reference in their entirety.Exemplary LNA analogs are described in U.S. Pat. Nos. 7,399,845 and7,569,686, both of which are hereby incorporated by reference in theirentirety.

In some cases (e.g., when the target nucleic acid is a miRNA, when thetarget sequence of the target nucleic acid spans a splice junction or afusion junction, etc.), the nucleic acid detection agent is an LNA.

—Modified Sugar Moieties—

A detection nucleic acid can also include one or more substituted sugarmoieties. Suitable polynucleotides include a sugar substituent groupselected from: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynylmay be substituted or unsubstituted C.sub.1 to C₁₀ alkyl or C₂ to C₁₀alkenyl and alkynyl. Also suitable are O((CH₂)_(n)O)_(m)CH₃,O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, andO(CH₂)_(n)ON((CH₂)_(n)CH₃)₂, where n and m are from 1 to about 10. Othersuitable polynucleotides include a sugar substituent group selectedfrom: C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkenyl, alkynyl,alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN,CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl,heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl,an RNA cleaving group, a reporter group, an intercalator, and othersubstituents having similar properties. A suitable modification caninclude 2′-methoxyethoxy (2′-O—CH₂ CH₂OCH₃, also known as2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim. Acta, 1995,78, 486-504) i.e., an alkoxyalkoxy group. A suitable modification caninclude 2′-dimethylaminooxyethoxy, i.e., a O(CH₂)₂ON(CH₃)₂ group, alsoknown as 2′-DMAOE, as described in examples hereinbelow, and2′-dimethylaminoethoxyethoxy (also referred to as2′-O-dimethyl-amino-ethoxy-ethyl or 2′-DMAEOE), i.e.,2′-O—CH₂—O—CH₂—N(CH₃)₂.

Other suitable sugar substituent groups include methoxy (—O—CH₃),aminopropoxy (—OCH₂ CH₂ CH₂NH₂), allyl (—CH₂—CH═CH₂), —O-allyl(—O—CH₂—CH═CH₂) and fluoro (F). 2′-sugar substituent groups may be inthe arabino (up) position or ribo (down) position. A suitable 2′-arabinomodification is 2′-F. Similar modifications may also be made at otherpositions on the oligomeric compound, particularly the 3′ position ofthe sugar on the 3′ terminal nucleoside or in 2′-5′ linkedoligonucleotides and the 5′ position of 5′ terminal nucleotide.Oligomeric compounds may also have sugar mimetics such as cyclobutylmoieties in place of the pentofuranosyl sugar.

—Base Modifications and Substitutions—

A detection nucleic acid may also include a nucleobase (also referred toas “base”) modifications or substitutions. As used herein, “unmodified”or “natural” nucleobases include the purine bases adenine (A) andguanine (G), and the pyrimidine bases thymine (T), cytosine (C) anduracil (U). Modified nucleobases include other synthetic and naturalnucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine,xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkylderivatives of adenine and guanine, 2-propyl and other alkyl derivativesof adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine,5-halouracil and cytosine, 5-propynyl (—C═C—CH₃) uracil and cytosine andother alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosineand thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino,8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines andguanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other5-substituted uracils and cytosines, 7-methylguanine and7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and3-deazaadenine. Modified nucleobases also include tricyclic pyrimidinessuch as phenoxazine cytidine(1H-pyrimido(5,4-b)(1,4)benzoxazin-2(3H)-one), phenothiazine cytidine(1H-pyrimido(5,4-b)(1,4)benzothiazin-2(3H)-one), G-clamps such as asubstituted phenoxazine cytidine (e.g.9-(2-aminoethoxy)-H-pyrimido(5,4-(b) (1,4)benzoxazin-2(3H)-one),carbazole cytidine (2H-pyrimido(4,5-b)indol-2-one), and pyridoindolecytidine (H-pyrido(3′,2′:4,5)pyrrolo(2,3-d)pyrimidin-2-one).

Heterocyclic base moieties may also include those in which the purine orpyrimidine base is replaced with other heterocycles, for example7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone.Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808,those disclosed in The Concise Encyclopedia Of Polymer Science AndEngineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons,1990, those disclosed by Englisch et al., Angewandte Chemie,International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y.S., Chapter 15, Antisense Research and Applications, pages 289-302,Crooke, S. T. and Lebleu, B., ed., CRC Press, 1993. Certain of thesenucleobases are useful for increasing the binding affinity of anoligomeric compound. These include 5-substituted pyrimidines,6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.5-methylcytosine substitutions have been shown to increase nucleic acidduplex stability by 0.6-1.2° C. (Sanghvi et al., eds., AntisenseResearch and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) andare suitable base substitutions, e.g., when combined with2′-O-methoxyethyl sugar modifications.

—Conjugates—

Another suitable modification of a subject nucleic acid involveschemically linking to the polynucleotide one or more moieties orconjugates. These moieties or conjugates can include conjugate groupscovalently bound to functional groups such as primary or secondaryhydroxyl groups. Conjugate groups include, but are not limited to,intercalators, reporter molecules, polyamines, polyamides, polyethyleneglycols, and polyethers. Suitable conjugate groups include, but are notlimited to, cholesterols, lipids, phospholipids, biotin, phenazine,folate, phenanthridine, anthraquinone, acridine, fluoresceins,rhodamines, coumarins, and dyes.

Conjugate moieties include but are not limited to lipid moieties such asa cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA,1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem.Let., 1994, 4, 1053-1060), a thioether, e.g., hexyl-S-tritylthiol(Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660, 306-309; Manoharanet al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), a thiocholesterol(Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), an aliphaticchain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al.,EMBO J., 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259,327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid,e.g., di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res.,1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain(Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), oradamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36,3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta,1995, 1264, 229-237), or an octadecylamine orhexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277, 923-937).

Signal Detection and Detection Devices

Aspects of the invention include detecting a signal resulting from thedetection of a target nucleic acid. Signal detection can be carried outusing any instrument (e.g., liquid assay device) that can measure thefluorescent, luminescent, light-scattering or colorimetric signal(s)output from the subject methods. In some embodiments, the signalresulting from the detection of a target nucleic acid is detected by aflow cytometer.

Aspects of the invention include a liquid assay device containing afixed, permeabilized, and rehydrated cellular sample containing anuclease inhibitor (e.g., RNase inhibitor) and a nucleic acid detectionagent (see above). In some embodiments, the liquid assay device furtherincludes a protein detection reagent (see above).

In some embodiments, the liquid assay device for evaluating a cellularsample for the presence of the target nucleic acid is a flow cytometer.As such, in some instances, the evaluation of whether a target nucleicacid is present in a cell of a cellular sample includes flowcytometrically analyzing the cellular sample. In flow cytometry, cellsof a cellular sample are suspended in a stream of fluid, which ispassed, one cell at a time, by at least one beam of light (e.g., a laserlight of a single wavelength). A number of detectors, including one ormore fluorescence detectors, detect scattered light as well as lightemitted from the cellular sample (e.g., fluorescence). In this way, theflow cytometer acquires data that can be used to derive informationabout the physical and chemical structure of each individual cell thatpasses through the beam(s) of light. If a signal specific to thedetection of a target nucleic acid is detected in a cell by the flowcytometer, then the target nucleic acid is present in the cell. In someembodiments, the detected signal is quantified using the flow cytometer.

Utility

The methods, devices, compositions and kits of the invention find use ina variety of different applications. Methods of the invention aremethods of evaluating cells of a cellular sample, where the targetnucleic acid may or may not be present. In some cases, it is unknownprior to performing the assay whether a cell of the cellular sampleexpresses the target nucleic acid. In other instances, it is unknownprior to performing the assay whether a cell of the cellular sampleexpresses the target nucleic acid in an amount (or relative amount,e.g., relative to another nucleic acid or relative to the amount of thetarget nucleic acid in a normal cell) that is greater than (exceeds) apredetermined threshold amount (or relative amount). In such cases, themethods are methods of evaluating cells of a cellular sample in whichthe target nucleic acid of interest may or may not be present in anamount that is greater than (exceeds) or below than a predeterminedthreshold. In some embodiments, the methods of the invention can be usedto determine the expression level (or relative expression level) of anucleic acid in one or more (or all) individual cell(s) of a cellularsample.

The methods of the invention can be used to identify a cell of a cellsample as aberrant or non-aberrant. For example, some mRNAs (and/ormiRNAs) are known to be expressed above a particular level, or relativelevel, (i.e., above a predetermined threshold) in aberrant cells (e.g.,cancerous cells). Thus, when the level (or relative level) of signal (asdetected using the subject methods) for a particular target nucleic acid(e.g., mRNA) of a cell of the cellular sample indicates that the level(or relative level) of the target nucleic acid is equal to or greaterthan the level (or relative level) known to be associated with anaberrant cell, then the cell of the cellular sample is determined to beaberrant. To the contrary, some mRNAs (and/or miRNAs) are known to beexpressed below a particular level, or relative level, (i.e., below apredetermined threshold) in aberrant cells (e.g., cancerous cells).Thus, when the level (or relative level) of signal (as detected usingthe subject methods) for a particular target nucleic acid of a cell ofthe cellular sample indicates that the level (or relative level) of thetarget nucleic acid is equal to or less than the level (or relativelevel) known to be associated with an aberrant cell, then the cell ofthe cellular sample is determined to be aberrant. Therefore, the subjectmethods can be used to detect and count the number and/or frequency ofaberrant cells in a cellular sample. Any identified cell of interest canbe isolated for further study.

In some instances, it is unknown whether the expression of a particulartarget nucleic acid varies in aberrant cells and the methods of theinvention can be used to determine whether expression of the targetnucleic varies in aberrant cells. For example, a cellular sample knownto contain no aberrant cells can be evaluated and the results can becompared to an evaluation of a cellular sample known (or suspected) tocontain aberrant cells.

In some instances, an aberrant cell is a cell in an aberrant state(e.g., aberrant metabolic state; state of stimulation; state ofsignaling; state of disease; e.g., cell proliferative disease, cancer;etc.). In some instances, an aberrant cell is a cell that contains aprokaryotic, eukaryotic, or viral pathogen. In some cases, an aberrantpathogen-containing cell (i.e., an infected cell) expresses a pathogenicmRNA or a host cell mRNA at a level above cells that are not infected.In some cases, such a cell expresses a host cell mRNA at a level belowcells that are not infected.

In embodiments that employ a flow cytometer to flow cytometricallyanalyze the cellular sample, evaluation of cells of the cellular samplefor the presence of a target nucleic acid can be accomplished quickly,cells can be sorted, and large numbers of cells can be evaluated. Gatingcan be used to evaluate a selected subset of cells of the cellularsample (e.g., cells within a particular range of morphologies, e.g.,forward and side-scattering characteristics; cells that express aparticular combination of surface proteins; cells that expressparticular surface proteins at particular levels; etc.) for the presenceor the level (or relative level) of expression of a target nucleic acid.

In some embodiments, the methods are methods of determining whether anaberrant cell is present in a diagnostic cellular sample. In otherwords, the sample has been obtained from or derived from an in vivosource (i.e., a living multi-cellular organism, e.g., mammal) todetermine the presence of a target nucleic acid in one or more aberrantcells in order to make a diagnosis (i.e., diagnose a disease orcondition). Accordingly, the methods are diagnostic methods. As themethods are “diagnostic methods,” they are methods that diagnose (i.e.,determine the presence or absence of) a disease (e.g., cancer,circulating tumor cell(s), minimal residual disease (MRD), a cellularproliferative disease state, viral infection, e.g., HIV, etc.) orcondition (e.g., presence of a pathogen) in a living organism, such as amammal (e.g., a human). As such, certain embodiments of the presentdisclosure are methods that are employed to determine whether a livingsubject has a given disease or condition (e.g., cancer, circulatingtumor cell(s), minimal residual disease (MRD), a cellular proliferativedisease state, a viral infection, presence of a pathogen, etc.).“Diagnostic methods” also include methods that determine the severity orstate of a given disease or condition based on the level (or relativelevel) of expression of at least one target nucleic acid.

In some embodiments, the methods are methods of determining whether anaberrant cell is present in a non-diagnostic cellular sample. Anon-diagnostic cellular sample is a cellular sample that has beenobtained from or derived from any in vitro or in vivo source, includinga living multi-cellular organism (e.g., mammal), but not in order tomake a diagnosis. In other words, the sample has been obtained todetermine the presence of a target nucleic acid, but not in order todiagnose a disease or condition. Accordingly, such methods arenon-diagnostic methods.

Compositions and Kits

Also provided are reagents, compositions and kits thereof for practicingone or more of the above-described methods. The subject reagents,compositions and kits thereof may vary greatly and can include any of: afixed, permeablized, and rehydrated cellular sample (e.g., including anuclease inhibitor, e.g., a RNase inhibitor); a probe set for detectinga target nucleic acid; a cellular fixation reagent; a cellularpermeabilization reagent; an aqueous cellular post-fixation reagent; anuclease inhibitor (e.g., an RNase inhibitor); a nucleic acid detectionagent or reagent (e.g., a nucleic acid detection agent including asignal producing system that includes a labeled nucleic acid probe); asignal amplification component (e.g., a branched nucleic acid); aprotein detection reagent (e.g., an antibody or a or binding fragmentthereof); buffers appropriate for sample stabilization, dilution,storage (i.e., storage buffer), washes (i.e., wash buffer), and/ordissolution; a stimulating agent (e.g., a stimulator); etc. The variouscomponents of the reagents, compositions and kits may be present inseparate containers, or some or all of them may be pre-combined into asingle reagent mixture or single container.

In some embodiments, a composition includes a fixed, permeablized, andrehydrated cellular sample with a nuclease inhibitor (e.g., an RNaseinhibitor). In some instances, the composition further includes aprotein detection reagent (see above), a stimulating agent, and/or anucleic acid detection agent (see above).

In some embodiments, a kit includes a cellular fixation reagent, acellular permeabilization reagent, an aqueous cellular post-fixationreagent; and a nuclease inhibitor (e.g., an RNase inhibitor). In someinstances, the kit further includes a protein detection reagent (seeabove), a stimulating agent, a nucleic acid detection agent (see above),and/or a buffer.

In addition to the above components, the subject kits may furtherinclude (in certain embodiments) instructions for practicing the subjectmethods. These instructions may be present in the subject kits in avariety of forms, one or more of which may be present in the kit. Oneform in which these instructions may be present is as printedinformation on a suitable medium or substrate, e.g., a piece or piecesof paper on which the information is printed, in the packaging of thekit, in a package insert, and the like. Yet another form of theseinstructions is a computer readable medium, e.g., diskette, compact disk(CD), flash drive, and the like, on which the information has beenrecorded. Yet another form of these instructions that may be present isa website address which may be used via the internet to access theinformation at a removed site.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Centigrade,and pressure is at or near atmospheric. In addition, common laboratoryprotocol abbreviations may be used (e.g., hr=hours, min=minutes,ml=milliliters, ul=microliters, rpm=revolutions per minute, g (in thecontext of centrifugation)=times the force of gravity, etc.).

Example 1

A single cell RNA flow (SCRF) methodology is presented that allowssensitive multiplexed co-detection of protein-based markers along withmRNA transcripts at a concentration of as few as one copy (e.g., as fewas 5 copies, as few as 10 copies, as few as 15 copies, etc.) per cell.

Materials and Methods

Primary Cells and Cell Lines

Human U937 cells were cultured in RPMI supplemented with 10% fetal calfserum (FCS). For stimulations, cells were incubated in culture mediumsupplemented with 50 ng/ml of phorbol myristate acetate (PMA) for 24hours. The B16 melanoma cell line stably transfected with mouse FMS-liketyrosine kinase 3 ligand (Flt3L) was a kind gift from Dr. EdgarEngleman, Stanford University. Flt3L-conditioned media was harvestedfrom these B16 melanoma cells after 3-5 days culture in RPMI 1640supplemented with 10% FCS, antibiotics, β-mercaptoethanol, and stored at−20° C. Concentration was determined by enzyme linked immunosorbentassay (ELISA) (antibody pairs purchased from R&D Systems). Primary bonemarrow cells were collected from femur, tibia, pelvis, and spine of 5-10week-old female BALB/c mice obtained from The Jackson Laboratory. Allanimal studies were done in compliance with the Stanford AdministrativePanel on Laboratory Animal Care Protocol 15986.

Isolation of Splenocytes and Bone Marrow Cells; Generation of BoneMarrow-Derived Flt3L Dendritic Cells

Splenocytes were isolated by dispersing the spleen through a 70-μm cellstrainer with the plunger from a 3-mL syringe in ice cold Hank'sBalanced Salt Solution (HBSS, Gibco). Red blood cells were lysed withACK lysis buffer (150 mM NH4Cl, 1 mM KHCO3, 100 uM EDTA in PBS), washedtwice, then resuspended in RPMI 1640 supplemented with 10% FCS,antibiotics, and β-mercaptoethanol. Bone marrow cells cultured withFlt3L (FLDCs) were prepared by crushing and flushing the femur, tibia,pelvis, and spine, using a 5-mL syringe tip, pouring the single cellsuspension through a 70-um cell strainer, lysing red blood cells withACK lysis buffer, washing twice, then resuspending at 1.5×10⁶ cells/mLin RPMI 1640 supplemented with 10% FCS, antibiotics, β-mercaptoethanol,and 15 ng/mL Flt3L (from conditioned media) for 8 days.

DC Enrichment, CpG Stimulation, and Cytokine Staining

Whole bone marrow or splenocytes were isolated as described above, andthen enriched with Miltenyi Biotec's Plasmacytoid Dendritic Cell Kit II.Dendritic cells (DCs) were stimulated at 10⁵ cells/mL, and wholesplenocytes were stimulated at 10⁷ cells/mL at 1 μM final concentrationof CpG-A 2216 (Invivogen). FLDCs were stimulated at 1.5×10⁶ cells/mLwith 1 μM final concentration of CpG-A 2336 (Invivogen) for 9-11 hoursat 37° C.; Brefeldin A (Ebioscience) was added after 5-7 hours. Afterstimulation, cells were pelleted, dead cells were stained with Live/DeadAqua (Invitrogen), and cells were fixed/permeabilized with Ebioscience'sFoxp3 kit. Cells were then stained with IgM-biotin, B220-PeCy7,CD11b-PercpCy5.5 (BD Biosciences), 120g8-PE, IFNα-FITC (clone RMMA-1,PBL Interferon Source), and TNF-700 (BD Biosciences) for 30 minutes onice, followed by a streptavidin-Qdot605 (Invitrogen).

SCRF (Single Cell RNA Flow) Protocol

When necessary, live cells were pre-stained for 30 min on ice with anantibody mix in PBS supplemented with 0.5% BSA or in RPMI supplementedwith 2% FCS. After staining, cells were washed twice with PBS, 0.5% BSAand fixed for 10-20 min at room temperature by adding formaldehyde up tofinal 4% directly to the staining medium. When antibody pre-staining wasnot performed, cells were fixed directly by adding formaldehyde up tofinal 4% directly to the culture medium. Following fixation, fixed cellswere pelleted by centrifugation at 600 g for 5 min and resuspended in atleast 10 ul residual volume by vortexing. Cells were placed on ice andpermeabilized by incubation in more than 20 residual volumes of ice-coldmethanol for 10 min. Following permeabilization, cells were stored at−80° C. in methanol.

Before the hybridization, cells were pelleted at 600 g for 5 min (allsubsequent spins were at this speed and duration), thoroughly vortexedin the residual volume, and post-fixed as follows: First, cells wereresuspended and incubated for 5 min in an ice-cold 1:1 methanol/4%formaldehyde in PBS, 0.1% Tween 20 (PBST). Cells were then pelletedagain and resuspended in 4% formaldehyde in PBST (For the post-fixationstep, either 4% or 10% formaldehyde in PBST was used, see FIG. 13).Following a 20 min incubation with rotation at room temperature, cellswere pelleted and washed twice with PBST and 1 U/ml RNasin. Finally,cells were resuspended in 1 volume of PBST and 3 volumes of ProteaseStop Buffer (Affymetrix QuantiGene ViewRNA kit). Cells were split intoaliquots (up to 5 million cells per 60 μl) and placed into hybridizationtubes.

Hybridizations were performed according to instructions supplied withthe QuantiGene ViewRNA kit. Briefly, 1.2 μl of probe in 60 μl ofProbe-Diluent were added to each tube. Samples were incubated either 3hours or overnight at 40° C. with shaking. Hybridizations were then spunand all but 30 μl of supernatant was removed. Cells were washed threetimes with 150 μl of Affymetrix Wash Buffer, each time leaving 30 μl ofvolume. After the last wash, 30 μl PreHybridization buffer, 1.2 μlPre-Amplifier, and 28.8 μl Amplifier Diluent were added to the 30 μl ofcells. After 2 hours at 40° C., 200 μl of Affymetrix Wash Buffer wasadded. After the solution was mixed thoroughly, cells were spun, andsupernatant was removed except for 30 μl. Cells were washed twice with150 μl of Affymetrix Wash Buffer, each time leaving 30 μl liquid.Following the second wash, 30 μl Pre-Hybridization buffer, 1.2 μlAmplifier, and 28.8 μl Amplifier Diluent were added to the 30 μl ofcells. After 2 hours of hybridization at 40° C., 200 μl of AffymetrixWash Buffer were added straight to the hybridization mixtures. After thesolution was mixed thoroughly, cells were spun, and the supernatant wasremoved leaving 30 μl of residual volume. Cells were washed twice with150 μl Affymetrix Wash Buffer, each time leaving 30 μl of residualvolume. After the last wash, 30 μl Pre-Hybridization buffer, 1.2 μllabeled probe (LP), and 28.8 μl Labeled Probe Diluent were added to the30 μl of cells. After 2 hours of hybridization at 40° C., samples werespun and all but 30 μl of the hybridization mix was removed. Cells werewashed twice with 150 μl Affymetrix Wash Buffer, each time leaving 30 μlof residual volume. If post-staining for protein markers was necessary,the cells were washed once with 150 μl PBST, pelleted, and stained in100 μl PBST with antibody. Cells were washed twice with PBST andanalyzed on an LSRII or a FACSAria (BD Bioscience).

Antibodies Used for Flow Cytometry and Cell Sorting

The following antibodies were purchased from BD Biosciences: pStat3(pY705), pStat5 (pY694), CD11a, CD36, QDot605-labeled anti-CD45, PacificBlue-labeled anti-CD3, FITC-labeled anti-CD34, PerCP-Cy5.5-labeledanti-CD33. Purified CD321 was purchased from eBiosciences. Conjugationof NHS-Ax488 and NHS-Ax647 dyes (Invitrogen) to purified antibodies wasperformed using protocols supplied by manufacturer. For sorting of blastcells, fixed, permeabilized, stained PBMCs were gated asSSC^(medium-high), CD45^(medium), CD3^(negative). Signaling subsets weresorted from gated blast cells based on pStat5 and pStat3 staining.CD3-positive T cells were sorted for RNA quality control.

Three Different Optional Steps

(1) Prior to fixation, live cells can be contacted with an antibody thatspecifically binds a surface marker (A labeled antibody can be used aslong as the label and antibody resist the subsequent methanoltreatment). To do this, live cells are contacted with ice cold antibodysolution for 30 minutes. The antibody solution contains the antibodydiluted in either (i) phosphate buffered saline (PBS) supplemented with0.5% bovine serum albumin (BSA) or (ii) RPMI supplemented with 2% fetalcalf serum (FCS). After contact with the antibody solution, the cellsare washed twice with same solution (minus the antibody). Proceed toFixation step.(2) After fixation and prior to permeabilization, cells can be contactedwith an antibody that specifically binds a cellular protein (A labeledantibody can be used as long as the label and antibody resist thesubsequent methanol treatment). To do this, fixed cells are pelleted bycentrifugation at 600 g for 5 minutes. Cells are then be re-suspended inresidual volume by vortexing. Cells are contacted with a solution of0.1% Saponin and 4% formaldehyde in PBS for 10 minutes at roomtemperature. Cell are centrifuged, followed by 3 washes in a solution of0.1% Saponin and RNas inhibitor (1/1000 dilution) in PBS at 4° C. Cellsare incubated for 5 minutes at room temperature in staining solution(minus antibody) supplemented with RNase inhibitor (1/50 dilution).Antibody is added and samples are incubated for 30 minutes at 4° C. withperiodic shaking. Three quick washes are performed with 1.5 ml of 0.1%Saponin supplemented with RNase inhibitor (1/500 dilution) in PBS.Solution is replaced with 0.1% Saponin supplemented with 4% formaldehydein PBS for 10 minutes at room temperature. Proceed to permeabilizationstep.(3) After the hybridization step used here, and prior to the flowcytometry detection step used here, the cellular sample can be contactedwith an antibody that specifically binds a cellular protein. To do this,cells are spun down via centrifugation, and resuspended and incubated inan antibody-containing solution of PBST and RNas inhibitor (diluted1/100) for 30 minutes at room temperature. Cells are then washed twicewith the same solution (minus the antibody). Proceed to flow cytometrystep.ResultsBranched DNA Cascades Allow Sensitive Detection of RNA by Flow Cytometry

RNA in situ hybridization is a well-established technique that producesreliable data in adherent cells and whole mount embryos of variousorganisms. Yet application of this technique in the context of flowcytometry (Mosiman et al. (2000) In Situ Hybridization in FlowCytometry, G. B. Faguet, Editor, Humana Press Inc.: Totowa, N.J. p.231-251) is rarely reported. In the course of initial experimentsperformed in this study extensive RNA degradation was observed whenfixed and permeabilized cells were moved to a water phase for subsequenthybridization or antibody-based staining (data not shown). In protocolswhere RNA in situ hybridization is followed by microscopy this step ispreceded by air-drying, which apparently aids in preserving the RNA. Theair-drying though is not compatible with flow cytometry. Using celllines and human PBMCs as models, a protocol was developed to inactivateRNases that remain active in fixed cells. Cells were first fixed andthen permeabilized by methanol. Importantly, following the methanol steppost-fixation by paraformaldehyde was performed. We have found that thisadditional fixation step allows recovery of intact RNA from fixed andstained cells. Post-fixation enables the paraformaldehyde basedinactivation of RNAses (Jonsson, N. et al. (1959) Histochemie, 1(4): p.251-256) in underfixed intracellular compartments rendered permeable bymethanol treatment. The reliability of this procedure is supported bycomparative microarray analysis that shows linear correlation ofexpression values obtained in live and fixed cells.

A combination of post-fixation based cell pretreatment and “branchedDNA” based fluorescent signal amplification (QuantiGene View RNA)(Piotrowska, J., et al., (2010) Journal of virology, 84(7): p. 3654-65;Taylor, A. M., et al., (2009) The Journal of neuroscience, 29(15): p.4697-707) herein called “SCRF” (Single Cell RNA Flow) was used forcytometric mRNA detection. Branched DNA signal amplification works byexponential build-up of a tree-like detection structure (FIG. 1A) suchthat every next probe of a cascade generates more templates for thesteps to follow. To examine the quantitative performance of the SCRFprotocol, a panel of target genes (GAPDH, PPIB, HPRT, POLR2A, HMBS)known to be constitutively expressed at different levels ranging fromthousands of copies of transcript per cell (GAPDH) (Bustin, S. A.,(2000) Journal of molecular endocrinology, 25(2): p. 169-93; Wilkening,S., et al. (2003) Drug metabolism and disposition: the biological fateof chemicals, 31(8): p. 1035-42) to ten (HPRT, PoIR2A) (Steen, A. M., etal., (1990) Experimental cell research, 186(2): p. 236-44), to fewer(HMBS) was assembled based on literature and database searches (Carter,M. G., et al., (2005) Genome Biology, 6(7): p. R61). For each gene, 10to 20 short antisense DNA probes incorporating a target-specific regionsand a dedicated sequence for proximity-based generation of a landingsite for the amplification cascade were synthesized. The detection oftranscripts in the panel was performed in U937 human cancer cell line.Branched DNA detection cascades were assembled by four sequentialhybridizations of fixed, permeabilized and postfixed U937 cells withdetection probes, preamplifier, amplifier and detector labeled with Ax(ALEXA FLUOR®) 647 dye as recommended in QuantiGene View RNA protocol.Application of the full cascade without addition of pre-amplifier probeswas used as a control. The hybridized samples were concurrently analyzedby both flow cytometry (FIG. 1B) and microscopy (FIG. 1D). Averagenumber of fluorescent signals per cells was estimated from confocalstacks collected for 100 cells for each type of transcript. As expectedfrom previous studies (Femino, A. M., et al. (2003) Methods inenzymology, 361: p. 245-304; Itzkovitz, S. et al. (2011) Nature Methods,8(4 Suppl): p. S12-9; Raj, A., et al., (2008) Nature Methods, 5(10): p.877), the number of fluorescent spots per cell agreed with the number oftranscripts per cell calculated based on absolute quantification by qPCR(data not shown) and reports in the literature (Pannetier, C., et al.,(1993) Nucleic Acids Research, 21(3): p. 577-83). The distribution ofthe flow cytometric signal from the least expressed gene HMBS (average 8transcripts per cell) was clearly different from control where the firststep probe was omitted. A linear correspondence between the relativemedium fluorescence intensity rMFI (rMFI=MFIgene/MFIcontrol) and theaverage number of spots-transcripts per cells was observed, indicatingthe wide dynamic range of the SCRF detection (FIG. 1C). Considering theability of flow cytometry to discern the signal with rMFI>2, weestimated that with Ax647 labeled detection cascade the lowest number oftranscript detectable by SCRF to be around 5 molecules per cell. Samepanel of genes was detected with branched DNA cascades labeled withother fluorophores (Ax 546, Ax488, Ax 750). With all the dyes it waspossible to detect the lowest expressed genes yet the best resolutionwas achieved with Ax647 labeled cascade.

Using SCRF to Map the Expression Domains of Hematopoietic TFs.

To examine the performance of SCRF for multiplexed transcript detection,subsets of lineage depleted mouse bone marrow were analyzed for PU.1 andGATA1 mRNA expression. These genes encode transcription factors involvedin regulation of the early hematopoietic lineage split into commonmyelo-erythroid and common granulocyte-monocyte-lymphoid progenitors(CMPs and GMLPs, respectively) (Iwasaki, H. et. al. (2007) Oncogene,26(47): p. 6687-6696). Lineage negative (lin⁻) bone marrow cells wereenriched by magnetic depletion; stained with antibodies against cKIT andSCA1; sorted into lin⁻cKIT⁻, lin⁻SCA1⁺cKIT⁺ (LSK), and lin⁻SCA1⁻cKIT⁺(LK) subsets (FIG. 4A); fixed; and treated according to the SCRFprotocol for simultaneous detection of PU.1 and GATA1 mRNAs withbranched DNA cascades labeled with Ax488 and Ax647 accordingly. Inconcurrence with prior reports (Arinobu, Y., et al (2007) Cell stemcell, 1(4): p. 416-427; Zhang, P., et al., (2000) Blood, October 15;96(8):2641-8; Nerlov, C., et al. (2000) Blood, April 15; 95(8):2543-51;Koschmieder, S., et al. (2005) International journal of hematology,81(5): p. 368-377), we observed an overall opposing pattern of PU.1 andGATA1 expression in LK cells (FIG. 4C, long identified as committedhematopoietic progenitors (Akashi, K., et al., (2000) Nature, 404(6774):p. 193-7). The LSK cells, which generally are believed to include mousehematopoietic stem cells (Osawa, M., et al., (1996) Science, 273(5272):p. 242-5; Spangrude, G. J. et al. (1998) Science, 241(4861): p. 58-62)showed co-expression of low levels of PU.1 and GATA1 mRNA (FIG. 4D).Such co-expression of master regulators opposing lineages is thought torepresent so-called “multilineage priming” phenomenon (Miyamoto, T., etal. (2002) Developmental cell, 3(1): p. 137-147; Miyamoto, T. et al.(2005) International journal of hematology, 81(5): p. 361-367; Laslo,P., et al., (2006) Cell, 126(4): p. 755-766). A small subset of LSKcells exhibited high levels of GATA1 expression (FIG. 4D, blue gate), inagreement with a previous analysis demonstrating that these cells areCMPs (Arinobu, Y., et al (2007) Cell stem cell, 1(4): p. 416-427). Tofurther co-examine PU.1 and GATA1 expression in LK subsets by microscopyin a separate experiment expression PU.1 and GATA1 mRNA was detected insorted LK cells with cascade labeled by Ax647 and Ax546 (a combinationof dyes which provided the highest resolution in SCRF). Four distinctsub-populations of LK cells defined by PU.1 and GATA1 mRNA expression(FIG. 2A) were further sorted and examined by confocal microscopy. Asexpected, cells that expressed high levels of PU.1 exhibited no GATA1staining (FIG. 2B, panel 1). With the exception of cells that expressedvery high levels of GATA1 (about 6% of the LK cells) (FIG. 2B, panel 4),GATA1-expressing cells expressed low levels of PU.1 transcript (FIG. 2B,panel 3). In addition, we detected a small population (4%) thatco-expressed the two transcripts at high levels (FIG. 2B, panel 2).

Pre-sorting the populations based on protein staining was not necessaryprior to SCRF analysis. Generally, protein staining could be performedat three steps during the protocol. For sensitive epitopes that did notsurvive the hybridization conditions cells had to be stained live andfixed after staining, in which case the surface staining survived wellthrough the course of several hybridizations (FIG. 5). Otherwiseintracellular epitopes could be stained after the post-fixation andbefore the hybridizations (data not shown) or after the hybridizations(see below). A number of surface markers (FcγRII/III, CD34, CD127, Flt3)used for classic delineation of the branches of early hematopoieticdifferentiation cascade were detected simultaneously with PU.1 and GATA1transcripts (FIG. 6, 7) using the prestaining of live lineage depletedbone marrow cells. In contrast to the data obtained with a fluorescentGATA1 reporter, which suggests that 35% of CMPs express GATA1 protein(Arinobu, Y., et al (2007) Cell stem cell, 1(4): p. 416-427), all CD34⁺cells in LK populations were found to be GATA1 mRNA negative (FIG. 6B).The Flt3^(high) subset of cKIT⁻ cells showed intermediate levels of PU.1(FIG. 6G), consistent with previous data indicating that these cells arecommon lymphoid progenitors (CLPs) (Arinobu, Y., et al (2007) Cell stemcell, 1(4): p. 416-427). A subset of LSK cells expressed high levels ofFlt3 (FIG. 6I), in accordance with early separation of Flt3⁺lymphoid-primed multipotent progenitors (LMPP) and Flt3⁻ CMPs(Adolfsson, J., et al., (2005) Cell, 121(2): p. 295-306). It has beensuggested that there is a distinct population of PU.1^(high) cellswithin the Flt3^(high) subset (Arinobu, Y., et al (2007) Cell stem cell,1(4): p. 416-427), but no evidence for this population was detected inthis analysis. In agreement with prior reports, CD16/32^(high) cells inthe LK population expressed the highest levels of PU.1 of the subsetsanalyzed (FIG. 7H).

A number of RNAs encoding important hematopoietic transcription factors(C/EBP, GFI1, GFI1 b, Pax5, E2A, MEIS, MYB, GATA2) were co-detected withPU.1 and GATA1 transcripts (FIGS. 7-12). There was a strong correlationbetween PU.1 and C/EBP levels (FIG. 7A-C) and between GATA1 and GFI1blevels (FIG. 7J-L) reflecting a direct regulatory connection (Kummalue,T. et al. (2003) Journal of Leukocyte Biology, 74(3): p. 464-70; Huang,D. Y., et al., (2004) Nucleic Acids Research, 32(13): p. 3935-46).Repression of PU.1 by GFI1 was previously demonstrated (Laslo, P., etal., (2006) Cell, 126(4): p. 755-766; Spooner, C. J., et al., (2009).Immunity, 31(4): p. 576-586), and yet curiously a correlation betweenPU.1 and GFI1 expression in cKIT⁻ and LK cells was detected (FIG. 9A,B). Both genes are thought to be activated by C/EBP, therefore suchdiscrepancy is due to a lag between the mRNA and the protein of GFI1which is sufficient to inhibit PU.1 mRNA expression. Most lin⁻ cellswere Runx2^(high) (FIG. 10I), and LK cells expressed high levels of MYB(FIG. 10H), reflecting the proliferative status of these “progenitor”cells.

Using the same approach, we evaluated the expression of genes known tobe involved in B cell development including EBF1, Id2, Pax5, andE2A/TCF3 (FIG. 11, 12). A positive correlation of E2A levels wasobserved with levels of both GATA1 and PU.1 expression (FIG. 11A-F),supporting the role of E2A in development of multiple hematopoieticlineages (Lin, Y. C., et al., (2010) Nature Immunology, 11(7): p. 635;Semerad, C. L., et al., (2009) Proceedings Of The National Academy OfSciences Of The United States Of America, 106(6): p. 1930-5). Inconcurrence with commonly accepted lineage classification framework,EBF1 and Pax5 were detected in the cells of the cKIT⁻ population (FIG.11G-L, 12A-F) with low levels of GATA1 and PU.1 expression (FIG. 5D). Inagreement with previous detection in both myeloid (Reizis, B (2010)Current opinion in immunology, 22(2): p. 206-211) and lymphoid (Quong,M. W., et al. (2002) Annual Review Of Immunology, 20: p. 301-22)lineages, Id2 was detected in cKIT⁻ cells with low levels of GATA1 andintermediate PU.1 expression (FIG. 12G-L)

Using SCRF to Study Cytokine Production by Activated pDC.

Some protein epitopes lost their antigenicity over the course of theseveral hybridizations composing the SCRF protocol; however, manyproteins were successfully evaluated after SCRF. Staining withantibodies against B220 and 120g8 performed after the SCRF procedure wasused to identify the population of plasmacytoid dendritic cells (pDCs)in primary mouse bone marrow as well as in cultures of bone marrow grownin the presence of Flt-3 ligand (FLDC) (FIG. 3A, D, G). Only a subset ofpDCs produce type I IFN (Scheu, S., et al. (2008) Proceedings Of TheNational Academy Of Sciences Of The United States Of America, 105(51):p. 20416-20421; Olshalsky and Fitzgerald-Bocarsly, (2005) Methods MolMed. 116:183-94.55). Accordingly we found that IFNα was highly expressedat mRNA and protein levels in a fraction of pDCs activated with CpG(FIG. 3E, I), which is commonly used as a mimic of viral infection. TNFmRNA and protein were detected in both pDC (not shown) and non-pDCsubsets (FIG. 3F) of activated FLDC cultures.

The IFNα mRNA has a short half-life (Cavalieri, R. L., et al., (1977).Proceedings Of The National Academy Of Sciences Of The United States OfAmerica, 74(10): p. 4415-9), and is the reason that IFNα transcriptswere detected in only a portion of cells that expressed IFNα protein(FIG. 3E). In contrast, in all cells in which TNF RNA was detected, TNFwas also detected at the protein level (FIG. 3F). Recently severalstudies have noted the small relative size of a population capable ofproducing type I IFNs even within a larger population of seeminglyhomogeneous cells (J Hu, S. I. et al. (2009). Biophysical Journal,97(7): p. 1984; Jianzhong Hu et al. (2011) PLoS One. February 8;6(2):e16614; Zhao, M., et al., (2012) PLoS biology, 10(1): p. e1001249).Some reports attribute this phenomenon to general features of basaltranscriptional mechanisms implicated in IFN production (J Hu, S. I. etal. (2009). Biophysical Journal, 97(7): p. 1984); one study showed thatthe heterogeneity of cells with respect to IFN production is largelyalleviated upon ectopic overexpression of IRF7 (Zhao, M., et al., (2012)PLoS biology, 10(1): p. e1001249). It has been suggested before thatIRF7 is produced at high basal levels in pDCs (Izaguirre, A., et al.,(2003) Journal of Leukocyte Biology, 74(6): p. 1125-1138). Using SCRFlevels of IRF7 and IFNα transcripts were co-determined in activated bonemarrow pDCs. Strong upregulation of IRF7 RNA was detected in allactivated pDCs, yet only a subset of cells with high IRF7 levels alsoexpressed IFN RNA (FIG. 3H, I).

Discussion

The single cell RNA flow (SCRF) method described in this study allowsflow cytometric multiplexed co-detection, in both immortalized andprimary cells, of proteins and mRNA transcripts. Two innovations allowedspecific and sensitive detection of mRNA by flow cytometry. The firstwas the addition of a second fixation after the initial“fixation/permeabilization” steps. This led to a dramatic reduction inthe activity of RNases that otherwise would have degraded mRNA and othertranscripts when cells are placed in aqueous buffer. The second key issignal amplification by branched DNA cascades. Protein staining can bedone before or after mRNA amplification. The SCRF method currentlyallows multiplexed branched chain DNA-based detection of up to four mRNAtranscripts (three were detected simultaneously in this study) togetherwith antibody-based protein detection. As more branched DNA cascades aredesigned the number of transcripts that can be detected simultaneouslywill be limited only by the number of fluorometrically separablefluorophores available (currently 15).

In this study, SCRF was used for visualization of regulatoryinteractions in various subsets of mouse bone marrow. An early decisionpoint in hematopoietic lineage progression is thought to rely on twoself-activating and mutually inhibiting transcription factors, PU.1 andGATA1 (Graf, T. and T. Enver, Forcing cells to change lineages. Nature,2009. 462(7273): p. 587-94). GATA1 establishes the identity of erythroidlineage. GATA1 expression is especially prominent inmegakaryocyte-erythroid progenitor cells (MEPs), whereas high PU.1levels are associated with differentiation along the myelomonocyticlineage. CMPs are most proximally defined as the GATA1⁺Flt3⁻CD34⁺ subsetof LSKs and further downstream as CD34⁺FcγR⁻ cells of the LK population.CMPs are able to differentiate both along erythroid and monocyticlineages. The bi-potency of CMP has been attributed to so-called“multilineage priming” state whereby the founder cells are able toco-express otherwise mutually exclusive lineage specific genes(Miyamoto, T., et al. (2002) Developmental cell, 3(1): p. 137-147;Miyamoto, T. et al. (2005) International journal of hematology, 81(5):p. 361-367). While GATA1 transcript levels were not tested in singlecell co-expression experiments around 30% of CMP were shown to expressGATA1 protein (Arinobu, Y., et al (2007) Cell stem cell, 1(4): p.416-427). Yet our data indicated that none of the CD34⁺ LK cells (asubset that contains the classically defined CMPs) express the GATA1transcript. This observation demonstrates that the “multilineage primed”state exists due to asynchrony between the protein and transcriptlevels. Specifically in the case of CMPs, which originate fromGATA1^(high) cells of the LSK compartment, the downstream “primed” statecorresponds to cells with induced CD34 expression that retain sufficientGATA1 protein for activity, but lack detectable GATA1 transcripts due toinhibition of GATA1 transcription and activation of CD34 expression byelevated levels of PU.1.

Mathematical modeling suggested that the behavior of PU.1-GATA1 systemis analogous to that of a bi-stable switch, with addition of anextra-stable state characterized by intermediate expression levels ofboth of the cross-inhibitory factors. It has been hypothesized thatlineage specification corresponds to transition of such a system from astate with three attractors to a state with two attractors; in thisstate only mutually exclusive expression patterns would be observed(Huang, S., et al., (2007) Developmental biology, 305(2): p. 695-713).Our data confirms the “multilineage primed” state of mouse LSKpopulation, where intermediate levels of cross-inhibitory PU.1 and GATA1were observed. Contrary to expected cancellation of this state incommitted progenitors, however, we observed a significant population ofcells co-expressing PU.1 and GATA1 within the LK cells. Interestingly,this population was largely confined to GATA1-expressing MEP cells,rather than to cells expressing high levels of PU.1. Notably, onaverage, there were no more then two fluorescent spots corresponding tothe PU.1 transcript in MEP cells co-expressing PU.1 and GATA1. In mostof the cells, at least one of these spots was located inside the nuclearperimeter; the other was proximal to nuclear periphery, but nuclearlocalization of this signal was not definitive. Thus, the PU.1 locus isactive in the majority of GATA1-positive MEP cells. Transcriptionalrepression of PU.1 by GATA1 relies on an interaction between PU.1 andGATA; this complex is unable to activate transcription (Nerlov, C., etal., (2000) Blood, April 15; 95(8):2543-51; Rekhtman, N. et al (1999)Genes Dev. un 1; 13(11):1398-411; Rekhtman, N. et al (2003) Molecularand Cellular Biology, 23(21): p. 7460). It is plausible that repressionof either gene could depend on its constant expression at levelssufficient to serve as a foundation for formation of repressivecomplexes. In such a scenario, minimal co-expression of GATA1 and PU.1should be observed in cells with high PU.1 or high GATA1. However, ourdemonstration of the complete lack of GATA1 expression in PU.1^(high)cells demonstrates the existence of specialized mechanisms of GATA1repression in PU.1^(high) LMPPs.

Use of RNA in situ hybridization has been instrumental in establishingthe “transcriptional bursting” model which has been recently widely usedto describe the heterogeneity (often manifested as bi-modality) in geneexpression observed in homogeneous cell populations (Munsky, B., G. etal. (2012). Science 336(6078): p. 183-187). The bursting paradigmaccommodates both expressional heterogeneity due to variable presence ofthe upstream activator (extrinsic bursting), and heterogeneity occurringeven in the constant presence of the activator (intrinsic bursting, sofar best explained by oscillatory behavior of the locus's permissivestate (Lionnet, T. and R. H. Singer, (2012) EMBO reports, 13(4): p.313-321). Most studies focusing on transcriptional bursting have beenperformed by manual transcript counting in individual cells. We usedSCRF cytometry to examine the heterogeneous production of IFNα byactivated plasmacytoid dendritic cells (pDC), which has been so fardescribed at the protein level. Previous studies indicate thattranscription of type I IFN genes is crucially dependent on the presenceof the transcription factor IRF7 (Honda, K., A. et al. (2006) Immunity,25(3): p. 349-360). It was shown that the expression of IFNb (Zawatzky,R., E. (1985) Proceedings Of The National Academy Of Sciences Of TheUnited States Of America, 82(4): p. 1136-40) and of other cytokines(Hollander, G. A., et al. (1999) Seminars in immunology, 11(5): p.357-67; Guo, L., et al. (2004) Immunity, 20(2): p. 193-203) is highlystochastic. A recent report from Maniatis' group (Zhao, M., et al.,(2012) PLoS biology, 10(1): p. e1001249) suggests that variability inIRF7 levels is one of the main reasons for the heterogeneous response inactivated cell cultures homogeneously capable of cytokine production. Wesought to explore IFNα production in pDCs and its dependence on IRF7.Surprisingly, only a minor subset of the population was capable of IFNαRNA synthesis despite high induction of IRF7 expression in all pDC. Thisobservation shows that additional mechanisms, not mediated by IRF7,account for the heterogeneity in IFN expression by pDCs. One suchmechanism is an oscillatory behavior of IFNα locus.

In this study the procedure developed for analysis of mRNA by flowcytometry was used to characterize regulatory gene expression indeveloping hematopoietic system and to analyze stochastic behavior of aparticular locus upon activation. As demonstrated, SCRF cytometry can beused for the detection of rare mRNAs, and finds application in analysisof clinically important cell subsets, such as virus-infected cells (HIVdiagnostics) or circulating tumor cells (cancer diagnostics). SCRFcytometry can also be used for evaluation of bioactive transcripts thatare not translated into proteins, such as of long non-coding RNAs. Widedynamic range and strong correlation between the average number ofmolecules and the relative MFI as detected by flow cytometry observed inour study shows that SCRF can be successfully employed for single-cellbased transcript counting in a multiplexed settings. These types of dataare currently obtained by manual transcript counting in a limited numberof cells. SCRF cytometry can also be used for analyses in whichantibodies necessary for protein expression studies are not available.

Example 2—Detection of miRNA (Micro RNA)

Methods

A methanol based buffer was used for permeabilization and fixation (thesame buffer used for mRNA (see Example 1) can also be used for miRNA).miRNA probe set design was similar to that used for the QG ViewRNA Cellassay (Affymetrix—Panomics). Reagents were used as in Example 1,including a methanol treatment, with the following details: one hourfixation with 4% PFA (as opposed to 10-20 minutes); one hourpost-fixation (as opposed to 20 minutes); sequential pre-amplificationand amplification hybridization was performed for 1.5 hours at 40° C.;the labeled probe (LP) was conjugated with alkaline phosphatase (AP) andhybridization was for one hour at 40° C.; Fast Red reagent (a substratefor AP) was used for signal detection; the Fast Red incubation was for30-45 min at 40° C.; and cells were fixed with 4% formaldehyde for 10min at room temperature after Fast Red incubation.

Results

A subject flow cytometry based analysis was performed to detect maturemiRNA in single cells using branched DNA signal amplification (FIG. 14).Multiple different miRNAs (Let7a, miR223, miR15a, and miR155) weredetected in human U937 cells (FIG. 15 and FIG. 16).

FIG. 15 demonstrates that the subject methods successfully detect miRNAsin human U937 cells. dapB (dihydrodipicolinate reductase) is a bacterialgene that is not present in human U937 cells and therefore serves as anegative control. (A) Let7a and miR223 were successfully detected usingflow cytometry. (B) miR15a and miR155 were successfully detected usingflow cytometry.

FIG. 16 demonstrates simultaneous detection of a miRNA (Let7a) and aprotein (beta-2 microglobulin; B2M) using branched DNA signalamplification (FIG. 14) and flow cytometry. In the top panel, B2M wasconjugated with a label moiety and the detected signal was light emittedat a peak wavelength of 488 nm. In the bottom panel, B2M was conjugatedwith a label moiety and the detected signal was light emitted at a peakwavelength of 750 nm signal. The Let7a signal was similar when onlyLet7a was detected (single plex) or when Let7a was detected incombination (duplex) with B2M. The B2M signal was reduced when detectedin duplex (in combination with Let7a) compared to when it was detectedin single plex. The reduction in B2M signal was likely due to the long(45 minute) Fast Red incubation. Regardless, B2M signal was clearlydetected in duplex with Let7a detection.

In summary, two experiments in U937 cells were performed and Let7adetection was reproduced consistently in both experiments (FIG. 15 andFIG. 16). miRNA targets successfully detected in U937 cells includeLet7a and miR223 (FIG. 15A); and miR155 and miR15a (FIG. 15B). Moreover,as demonstrated here via the detection of mature miRNA targets, thesubject methods can be used to detect target nucleic acids with shorttarget sequences. Thus, the subject methods are also useful for thedetection other target nucleic acids with short target sequences (e.g.,fusion gene transcripts, splice variants, etc.). In addition, it ispossible to simultaneously detect multiple different nucleic acidtargets (e.g., mRNA, miRNA, fusion gene transcripts, splice varianttranscripts, etc.) and proteins in a cell (FIG. 16) using the subjectmethods (e.g., using branched nucleic acid signal amplification and flowcytometry).

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is readily apparent to those of ordinary skill in theart in light of the teachings of this invention that certain changes andmodifications may be made thereto without departing from the spirit orscope of the appended claims.

Accordingly, the preceding merely illustrates the principles of theinvention. It will be appreciated that those skilled in the art will beable to devise various arrangements which, although not explicitlydescribed or shown herein, embody the principles of the invention andare included within its spirit and scope. Furthermore, all examples andconditional language recited herein are principally intended to aid thereader in understanding the principles of the invention and the conceptscontributed by the inventors to furthering the art, and are to beconstrued as being without limitation to such specifically recitedexamples and conditions. Moreover, all statements herein recitingprinciples, aspects, and embodiments of the invention as well asspecific examples thereof, are intended to encompass both structural andfunctional equivalents thereof. Additionally, it is intended that suchequivalents include both currently known equivalents and equivalentsdeveloped in the future, i.e., any elements developed that perform thesame function, regardless of structure. The scope of the presentinvention, therefore, is not intended to be limited to the exemplaryembodiments shown and described herein. Rather, the scope and spirit ofpresent invention is embodied by the appended claims.

What is claimed is:
 1. A method of assaying a cellular sample thatcomprises one or more cells in suspension for the presence of a targetnucleic acid, the method comprising: contacting the cellular samplewith, in order: (i) a fixation reagent; (ii) a permeabilization reagent;(iii) an aqueous fixative comprising one or more fixation reagentsselected from the group consisting of: a cross-linking fixative, aprecipitating fixative, an oxidizing fixative, and a mercurial fixative;and (iv) a nucleic acid detection agent comprising a signal producingsystem that comprises a branched nucleic acid signal amplificationcomponent, to produce a suspended hydrated/fixed detectionagent-contacted cellular sample; and measuring a signal produced by thesignal producing system to evaluate the suspended hydrated/fixeddetection agent-contacted cellular sample for the presence of the targetnucleic acid.
 2. The method according to claim 1, wherein the targetnucleic acid is a ribonucleic acid (RNA).
 3. The method according toclaim 2, wherein the RNA is a microRNA, a fusion gene transcript, or asplice variant.
 4. The method according to claim 1, wherein the methodfurther comprises contacting the cellular sample with a proteindetection reagent.
 5. The method according to claim 4, wherein theprotein detection reagent comprises a labeled binding member thatspecifically binds to a target protein.
 6. The method according to claim5, wherein the labeled binding member comprises an antibody or bindingfragment thereof.
 7. The method according to claim 6, wherein the labelis a metal.
 8. The method according to claim 6, wherein the measuringcomprises measuring the amount of label using mass cytometry.
 9. Themethod according to claim 1, wherein the signal producing systemcomprises a nucleic acid probe comprising a label that produces thesignal.
 10. The method according to claim 9, wherein the label is ametal.
 11. The method according to claim 10, wherein the measuringcomprises mass cytometry.
 12. The method according to claim 1, whereinthe measuring comprises flow cytometry.
 13. The method according toclaim 1, wherein the method comprises, after contacting the cellularsample with the aqueous fixative, storing the cellular sample for aperiod of time prior to the measuring step.
 14. The method according toclaim 13, wherein the storing is in an aqueous buffer comprising anuclease inhibitor.
 15. The method according to claim 14, wherein thestoring is at a temperature below 0° C.
 16. The method according toclaim 1, comprising contacting the cellular sample with a stimulatingagent prior to contact with the fixation reagent.
 17. The methodaccording to claim 1, wherein the method further comprises obtaining thecellular sample.
 18. The method according to claim 1, wherein thecellular sample is a human cellular sample.
 19. The method according toclaim 18, wherein the method further comprises employing a result of theevaluating step to diagnose a state of the human.
 20. The methodaccording to claim 18, wherein the state is a disease state.
 21. Themethod according to claim 20, wherein the disease state is a cellularproliferative disease state.
 22. The method according to claim 1,further comprising contacting the cellular sample with a nucleaseinhibitor after step (iii).
 23. The method according to claim 1, whereinthe permeabilization reagent comprises methanol.
 24. The methodaccording to claim 1, wherein the aqueous fixative comprises an aldehydefixative.
 25. The method according to claim 1, wherein the aqueousfixative comprises at least one of: formaldehyde and glutaraldehyde. 26.A method comprising: contacting a cellular sample that comprises one ormore cells in suspension with, in order: (i) a fixation reagent; (ii) apermeabilization reagent; (iii) an aqueous fixative comprising one ormore fixation reagents selected from the group consisting of: across-linking fixative, a precipitating fixative, an oxidizing fixative,and a mercurial fixative; to produce a hydrated/fixed suspended cellularsample.
 27. The method according to claim 26, wherein the method furthercomprises contacting the cellular sample with a protein detectionreagent.
 28. The method according to claim 27, wherein the proteindetection reagent comprises a labeled binding member that specificallybinds to a target protein.
 29. The method according to claim 28, whereinthe labeled binding member comprises an antibody or binding fragmentthereof.
 30. The method according to claim 26, wherein the methodfurther comprises contacting the cellular sample with a stimulatingagent.
 31. The method according to claim 26, wherein the cellular sampleis a human cellular sample.
 32. The method according to claim 26,further comprising contacting the cellular sample with a nucleaseinhibitor after step (iii).
 33. The method according to claim 26,wherein the permeabilization reagent comprises methanol.
 34. The methodaccording to claim 26, wherein the aqueous fixative comprises analdehyde fixative.
 35. The method according to claim 26, wherein theaqueous fixative comprises at least one of: formaldehyde andglutaraldehyde.
 36. The method according to claim 26, wherein thepermeabilization reagent comprises methanol.
 37. A method of assaying acellular sample that comprises one or more cells in suspension for thepresence of a target nucleic acid, the method comprising: contacting thecellular sample with, in order: (i) a first aqueous fixative; (ii) apermeabilization reagent; (iii) a second aqueous fixative comprising oneor more fixation reagents selected from the group consisting of: across-linking fixative, a precipitating fixative, an oxidizing fixative,and a mercurial fixative; and (iv) a nucleic acid detection agentcomprising a signal producing system that comprises a branched nucleicacid, to produce a suspended hydrated/fixed detection agent-contactedcellular sample; and measuring a signal produced by the signal producingsystem to evaluate the suspended hydrated/fixed detectionagent-contacted cellular sample for the presence of the target RNA,wherein the measuring comprises flow cytometry.
 38. The method accordingto claim 37, wherein the first and/or second aqueous fixative comprisesan aldehyde fixative.
 39. The method according to claim 37, wherein thefirst and/or second aqueous fixative comprises at least one of:formaldehyde and glutaraldehyde.
 40. The method according to claim 37,further comprising contacting the cellular sample with a nucleaseinhibitor after step (iii).
 41. The method according to claim 37,wherein the permeabilization reagent comprises methanol.
 42. The methodaccording to claim 1, wherein the cross-linking fixative is animidoester or a N-Hydroxysuccinimide (NHS) ester.
 43. The methodaccording to claim 26, w wherein the cross-linking fixative is animidoester or a N-Hydroxysuccinimide (NHS) ester.
 44. The methodaccording to claim 37, wherein the cross-linking fixative is animidoester or a N-Hydroxysuccinimide (NHS) ester.
 45. The methodaccording to claim 1, wherein the precipitating fixative is an alcohol,acetic acid, or acetone.
 46. The method according to claim 26, whereinthe precipitating fixative is an alcohol, acetic acid, or acetone. 47.The method according to claim 37, wherein the precipitating fixative isan alcohol, acetic acid, or acetone.